nitrogen fixation in acacias Many a tree is found in the wood, And every tree for its use is good; Some for the strength of the gnarled root, Some for the sweetness of fl ower or fruit.

Henry van Dyke, Salute the Trees

He that planteth a tree is the servant of God, He provideth a kindness for many generations, And faces that he hath not seen shall bless him.

Henry van Dyke, Th e Friendly Trees Nitrogen Fixation in Acacias: an Untapped Resource for Sustainable Plantations, Farm Forestry and Land Reclamation

John Brockwell, Suzette D. Searle, Alison C. Jeavons and Meigan Waayers

Australian Centre for International Agricultural Research 2005 Th e Australian Centre for International Agricultural Research (ACIAR) was established in June 1982 by an Act of the Australian Parliament. Its mandate is to help identify agricultural problems in developing countries and to commission collaborative research between Australian and developing country researchers in fi elds where Australia has a special research competence.

Where trade names are used, this constitutes neither endorsement of nor discrimination against any product by the Centre.

aciar monograph series Th is series contains results of original research supported by ACIAR, or deemed relevant to ACIAR’s research objectives. Th e series is distributed internationally, with an emphasis on developing countries.

© Australian Centre for International Agricultural Research 2005

Brockwell, J., Searle, S.D., Jeavons, A.C. and Waayers, M. 2005. Nitrogen fi xation in acacias: an untapped resource for sustainable plantations, farm forestry and land reclamation. ACIAR Monograph No. 115, 132p.

1 86320 489 X (print) 1 86320 490 3 (electronic)

Editing and design by Clarus Design, Canberra Foreword

Acacias possess many useful attributes — they are Over the past two decades, Australian scientists adapted to a wide range of warm-temperate and and their counterparts in partner countries have tropical environments including arid and saline sites, pursued the domestication of acacias through a and infertile and acid soils. Th e species most used in wide range of studies. Important outcomes include a forestry come from Australia or nearby countries, greater understanding of both the acacias and their but others in cultivation are from India, Myanmar, symbiotic micro-organisms, and the development of Arabia, Africa, tropical America and Hawaii. techniques for exploiting effi cient symbioses. Th ese developments are summarised in this review. Although acacias have been an important element of agricultural systems in Africa for centuries, more We compliment the authors of this publication. extensive cultivation commenced after Australian Th eir account is a valuable compilation of widely species were introduced into India for fuelwood dispersed information, enhanced by their capacity around 1850, and later to southern Africa for to assess its merit and relevance. Th ey conclude tanbark production. Th is latter role subsequently that improved nitrogen fi xation is a potential diff used, on a smaller scale, to other countries bonus whenever acacia is cultivated, and that including Brazil and China. the magnitude of the bonus will depend on both eff ective inoculation and good silviculture. Some hundred years later, the cultivation of acacias has again blossomed. Tropical species are attracting ACIAR is pleased to continue its strong support for attention for their potential to provide wood for the domestication of these important trees through industrial and domestic purposes, and the use of a the publication of this review. Th e publication is also variety of species for land rehabilitation, especially available on ACIAR’s website at . in Australia, is expanding rapidly.

Th e ability of legumes to fi x atmospheric nitrogen effi ciently has been exploited with conspicuous success in agriculture — pastures based on Peter Core, Director, Australian Centre for subterranean clover are a notable Australian International Agricultural Research example. Such successes, and the prominence of acacias in many natural ecosystems, have fuelled hopes that eff ective nitrogen fi xation by cultivated acacias would enhance the growth of both the acacia Alan Brown, Honorary Research Fellow, CSIRO and associated crops. Forestry and Forest Products

5

Contents

Foreword ...... 5 Dedication ...... 9 Preface ...... 11 Acknowledgments ...... 13

Abstract ...... 15

1. Introduction ...... 17

2. Th e plant ...... 18 Utilisation ...... 19 Distribution ...... 24 Plant taxonomy ...... 24 Root and nodule morphology ...... 28

3. Th e bacteria ...... 29 Acacia rhizobia in nature ...... 29 Taxonomy of acacia rhizobia ...... 31 Diversity of acacia rhizobia ...... 34 Comment on the taxonomy and diversity of acacia rhizobia ...... 36 Root infection and nodule formation ...... 36 Rhizobial products ...... 37

7 nitrogen fixation in acacias

4. Th e symbiosis ...... 39 Measurement of nitrogen fi xation ...... 41 Consideration of procedures for measuring N fi xation ...... 43 Nitrogen fi xation in glasshouse and nursery ...... 44 Nitrogen fi xation in the fi eld ...... 45 Environmental factors aff ecting nitrogen fi xation in the fi eld ...... 49 Mycorrhizal factors aff ecting nitrogen fi xation in the fi eld ...... 58 Rhizobial factors infl uencing nitrogen fi xation in the fi eld ...... 60

5. Enhancement of the symbiosis ...... 69 Th e need for inoculation ...... 69 Responses to rhizobial inoculation of acacias in fi eld and nursery ...... 69 Inoculants ...... 72 Breeding and selection for enhanced acacia symbiosis ...... 77

6. Exploitation of the symbiosis ...... 78 Natural exploitation ...... 78 General exploitation ...... 79 Intercropping ...... 79 Land reclamation: principles and practice — the Australian experience ...... 82

7. General conclusions and prognosis ...... 89 Th e plant ...... 89 Th e bacteria ...... 90 Th e symbiosis ...... 90 Exploitation ...... 91

References ...... 94

Appendix ...... 127

8 Dedication

Th is review is dedicated to the memory of Yvonne at the time of his death, was making meaningful Barnet, Alan Gibson and Ben Bohlool. Each of progress towards unravelling its complexities. these researchers was a pioneer in the fi eld of Professor Ben Bohlool was Director of the University nitrogen fi xation in the genus Acacia, each was a of Hawaii’s NifTAL Project (Nitrogen Fixation by distinguished scholar and teacher, and each died Tropical Agricultural Legumes), located at Paia, early and in tragic circumstances in the 1990s. Maui, Hawaii, a centre famous for its international training courses and workshops. Ben Bohlool’s Dr Yvonne Barnet, working at the University of New boundless enthusiasm instilled a lifelong interest South Wales, was among the very fi rst to recognise in nitrogen fi xation in literally hundreds of people. the extremes of morphological diversity within Many of them have added to current knowledge of acacia root-nodule bacteria. Her observation was the acacia symbiosis. one of the catalysts that eventually led to extensive taxonomic reclassifi cation of the Rhizobiaceae. Early During their lifetimes, Yvonne Barnet, Alan in his career, Dr Alan Gibson, CSIRO Plant Industry, Gibson and Ben Bohlool each made substantial Canberra, conducted ground-breaking research on contributions to research on legume nitrogen the physiological eff ects of environmental variables fi xation and the rhizobial symbiosis of the genus on the effi ciency of legume nitrogen fi xation. Later Acacia. Without them, our understanding of the in life, he developed an interest in host/plant fi eld would be diminished. We deeply regret their specifi city in the Acacia/rhizobia symbiosis and, passing.

9 Authors

john brockwell CSIRO Plant Industry GPO Box 1600 Canberra, ACT 2601 Australia

suzette d. searle PO Box 6201 O’Connor, ACT 2602 Australia Formerly of CSIRO Forestry and Forest Products

alison c. jeavons and meigan waayers Department of Primary Industries PO Box 3100 Bendigo Delivery Centre, Victoria 3554 Australia

10 Preface

It is a curiosity that, in Australia with its great Eff ectiveness and persistence of Acacia rhizobia’, wealth of native plants, so little science apart from when one of us (John Brockwell) was thrown in at taxonomy has been devoted to this unique fl ora. the deep end of the fi eld following the untimely Th is observation applies particularly to studies of death of Alan Gibson, a principal investigator for the nitrogen-fi xing symbiosis between Australian the subproject. A fi rst step in the learning process native legumes and their root-nodule bacteria. was a review of the literature. Th is turned out to be Th is is all the more strange when it is considered more substantial than we had imagined and highly that, as early as the late 1950s, Rob Lange, as dispersed in terms of the species investigated, a postgraduate student in the late Lex Parker’s the aspects of the symbiotic relationship studied laboratory at the University of Western Australia and the places of publication of results. Th ere in Perth, began publishing fascinating, thought- appeared to be three main reasons for this: fi rst, provoking papers on the diversity of legumes and the genus is so large — some 1350 species; second, root-nodule bacteria indigenous to the soils of it is more diffi cult to experiment with trees and the south-west of Western Australia. Of course, shrubs than it is with herbs; and third, it has been the fi eld has not been completely neglected. Ann unfashionable until recent times to work with Lawrie in Melbourne and the late Yvonne Barnet nitrogen-fi xing trees. It then occurred to us that in Sydney maintained an ongoing interest and the it would be helpful to our incomplete but growing occasional paper from other Australian sources has understanding of the fi eld if we were to bring the appeared in the literature from time to time. In literature together in the form of a review. Th is addition, the Australian Centre for International monograph is the result. We hope it will be useful to Agricultural Research (ACIAR) has sponsored several wider audiences. Our diff erent areas of experience international conferences to deal with manifold have been conducive to a fruitful, complementary aspects of the utilisation of Acacia species around collaboration. John Brockwell has worked for the world; from time to time, papers about the many years on a number of aspects of symbiotic acacia symbiosis have appeared in the proceedings. nitrogen fi xation in crop and forage legumes in agricultural settings and the utilisation of fi xed Th is monograph arose from an ACIAR project, nitrogen in phase-farming systems. Suzette Searle’s ‘Australian acacias for sustainable development extensive dealings with Australian acacias have in China, Vietnam and Australia — Subproject B. been directed at distribution, intraspecifi c variation,

11 nitrogen fixation in acacias climatic, topographic and edaphic adaptation, and is fi xed on Earth. Yet, the potential for exploiting utilisation for farm and plantation forestry in both acacia nitrogen fi xation has been almost completely Australia and Asia. Alison Jeavons and Meigan overlooked. We hope that this monograph will Waayers are working at the forefront of sustainable stimulate interest in tapping into this neglected land reclamation and are using acacias and other resource. Australian native trees and shrubs, and various establishment techniques, for both large- and small- At times, we have been emphatic and contentious. scale revegetation of damaged lands. Th at has been deliberate and we don’t apologise. It has been our aim to stimulate discussion and ideas. Th ere is, of course, a plethora of literature dealing If we can encourage even a small number of people with legume nitrogen fi xation. Nonetheless, we feel to interest themselves in, and perhaps study, the that the appearance of this monograph is timely. hugely diverse and fascinating genus that is Acacia, At the time of writing the preface (mid-2004), its equally diverse root-nodule bacteria and their world crude oil prices have risen to record levels, capacity together to fi x nitrogen from the infi nite reminding us once again, if that were ever necessary, resource of the Earth’s atmosphere, then we will that fossil fuels and their nitrogenous fertiliser by- have achieved our objective. As for ourselves, we products are a fi nite resource. Anything that can enjoyed the experience of compiling the monograph. be done to utilise nitrogen biologically fi xed from Our review of the literature has taken us into quite the atmosphere to replace or supplement the use new terrain. We cannot help but admire those of fertiliser nitrogen must be of ultimate benefi t to people who have made observations and conducted the human race and the environment. Estimates of fi eld experiments, some quite sophisticated, the amounts of legume nitrogen fi xed worldwide in diffi cult circumstances and in remote and go as high as 100 million tonnes annually. Acacias sometimes dangerous places and, in so doing, represent 6–7% of the more than 20,000 known started to unravel the complexities of the symbiosis species of legumes and must make a very substantial between Acacia and its root-nodule bacteria. contribution to the total amount of nitrogen that

12 Acknowledgments

Many people helped us, in small ways and large, in production unit’ for inoculant preparation, to compiling the monograph; we mention here only Gary Bullard, formerly Bio-Care Technology Pty a few of them. We are greatly indebted to Mike Ltd, Somersby, New South Wales, for providing Trinick, formerly CSIRO Plant Industry, Canberra, a manufacturer’s perspective of essential for allowing us to make use of his database of characteristics of good legume-inoculant strains, historical literature references to acacia symbioses, and to Jackie Nobbs, SARDI, Adelaide, for providing and to Michelle Hearn, CSIRO Black Mountain the authority for the nematode name, Meloidogyne Library, Canberra, for her patience in unearthing javanica. We thank Sandra McIntosh and Siobhan obscure references. Partap Khanna, CSIRO Forestry Duff y, CSIRO Plant Industry Visual Resources and Forest Products, Canberra, kindly allowed us to Unit, Canberra, for preparing the illustrations. In use some of his unpublished data on relative growth particular, we are most grateful to Mark Peoples, rates of Acacia and Eucalyptus growing in mixed CSIRO Plant Industry, Canberra, for allowing us stands that showed how the eucalypts responded to make use of some of his unpublished results, to to the presence of acacias that were actively fi xing Janet Sprent, University of Dundee, UK, for very nitrogen. In this same context, we are pleased to helpful discussions and her encouragement, and to acknowledge the editor of Th e Canberra Times, Alan Brown, CSIRO Forestry and Forest Products, a newspaper published daily in Canberra, ACT, Canberra, and Jeremy Burdon, CSIRO Plant Australia, for permission to reproduce a cartoon Industry, Canberra, for their critical appraisals of relating to the benefi ts of growing species of Acacia the manuscript. and Eucalyptus in mixtures. We are indebted to Janet Sprent, University of Dundee, UK, Ken Th e monograph contains a number of photographs. Giller, University of Wageningen, Th e Netherlands, Less than half of them are our own. We are and Margaret Th ornton, CSIRO Black Mountain especially indebted to those people who allowed us Library, Canberra, for assisting us in our eff orts to access to their fi les of photographs and generously keep abreast of the recent rapid developments in gave us permission to reproduce them. Sources are the taxonomy of the legume root-nodule bacteria. acknowledged with the photographs. We are grateful also to Paul Singleton and the NifTAL Center and MIRCEN, Paia, Hawaii, USA, In compiling this review, it was never our intention for supplying us with details of NifTAL’s ‘micro- to have an exhaustive list of citations to the

13 nitrogen fixation in acacias nitrogen-fi xing symbiosis in the genus Acacia. It addition, we take full responsibility for other errors is certain, however, that we must have overlooked and omissions and will be grateful to whomsoever some works of substance that should have been draws them to our attention. included. We apologise to the authors concerned. In Alison Jeavons Acacia paradoxa

14 Abstract

Th e legume genus Acacia has some 1350 species and Factors that might limit nitrogen fi xation are is distributed throughout the world, particularly in considered, with the conclusion that, as with other Africa, Asia and Australia. Th e genus is exploited legumes, nitrogen-fi xing ability is best expressed in in natural habitats and plantations for many the absence of limiting factors, especially defi ciencies purposes. It forms a symbiotic association with of nutrients and soil moisture. Th ere is usually a strains of at least six genera of root-nodule bacteria diversity of strains of rhizobia in soils where acacias (rhizobia) that are also widely distributed. Many of grow naturally. Many of these strains do not nodulate the associations fi x nitrogen from the atmosphere, Acacia spp. at all and many others that do form but there is great variation in nitrogen-fi xing nodules have little or no capacity to fi x nitrogen. specifi city in both hosts and bacteria — some acacias However, it appears that, within the total population fi x nitrogen with only a small number of rhizobial of naturally occurring rhizobia, there are invariably strains, others are more promiscuous. present at least some strains that are capable of fi xing signifi cant amounts of nitrogen in association Records indicate that acacias fi x less nitrogen than with acacias. Th ere is no convincing evidence that, other leguminous trees. However, this impression in natural environments, non-infective, ineff ective appears to be an artefact of the ecosystems where or poorly eff ective rhizobia themselves are ever a the measurements were made. Most assessments constraint on acacia nitrogen fi xation. of acacia nitrogen fi xation have been undertaken in forests or woodlands where nitrate in the soils We conclude that little can be done to enhance often inhibits nitrogen fi xation, whereas the acacia nitrogen fi xation in forests or established nitrogen fi xed by other tree legumes has usually plantations except, where economically feasible, been measured in anthropogenic ecosystems such to correct nutrient imbalances and to control as plantations, hedgerows and coppices, where soil pests. On the other hand, there appears to be great nitrate is less inhibitory. We conclude that acacias and inexpensive scope to use inoculation with have the capacity to fi x useful quantities of nitrogen eff ective strains of rhizobia to improve the vigour but that, unlike the plant itself, its symbiosis is and nitrogen fi xation of seedlings grown in plant under-utilised. nurseries as tube stock destined for outplanting into the fi eld. Where outplantings are made, inoculated,

15 nitrogen fixation in acacias well-nodulated seedlings survive better and grow In conclusion, there are compelling arguments faster than their uninoculated counterparts. that acacia nitrogen fi xation can be far better Inoculation with rhizobial strains cultured in a peat exploited than it has been in the past. Th is carrier, using a procedure termed soil enrichment, will involve eff ective rhizobial inoculation of is postulated as an effi cient means of producing seedling stock in nurseries and development of vigorous, well-nodulated, nitrogen-fi xing acacia methods of inoculation of acacia seed intended seedlings in nurseries. Implications for commercial for surface seeding. Th ere seems no doubt that, manufacture of acacia inoculants are discussed. properly exploited, the symbiosis has the capacity to contribute to the productivity of acacia Th ree factors are especially relevant to the timeliness and companion species in plantations, to the and signifi cance of this review: (i) the already rehabilitation of eroded and salinised lands, and substantial, and expanding, scale of acacia plantings to the augmentation of reserves of nitrogen in the in plantations and on farms, (ii) the potential soil. Even in circumstances where inoculation is of diverse Acacia species for the reclamation of not practicable, the cultivation of acacias has the degraded landscapes, and (iii) the expanded pool of potential to enhance soil fertility and soil structure. research results relating to acacias generally that has accumulated over the past 15 years. Alison Jeavons Acacia podalyriifolia

16 1. Introduction

Th e world uses huge quantities of synthetic fi xation by acacias to benefi t their productivity and nitrogenous fertiliser for growing plants. Th is the sustainability of the natural and anthropogenic dependence creates certain dangers for the global ecosystems in which they grow. economy and especially for the environment (Smil 1997). Anything that can be done to utilise nitrogen We consider fi rst some literature relating (N) fi xed naturally from the atmosphere — where it independently to the plant and its root-nodule occurs as molecular nitrogen (N₂) — as a substitute bacteria, then deal with the symbiotic association for fertiliser N, will benefi t all people. between them. We observe that, in many respects, the processes of N fi xation in acacias are similar to Acacia species are legumes and, in symbiotic those that apply to legumes generally. Th erefore, association with root-nodule bacteria, are when we could fi nd no literature relevant to acacias, partners in fi xation of atmospheric N (N fi xation). we have drawn upon information for other legumes. Estimation of the total quantity of legume N fi xed Finally, we speculate on how the acacia symbiosis worldwide is an exercise in informed guesswork, might be exploited. but the amount is of the order of 70–100 million tonnes annually. Since the known number of Acacia species represents some 6–7% of the 20,000 species of legumes, acacias must make a substantial contribution to the total quantity of N fi xed in terrestrial natural systems.

Duke (1981) lists six species of Acacia in his ‘Handbook of legumes of world economic importance’ and many others are utilised in a

multiplicity of ways by humans and all types of Brockwell John animals. However, little eff ort has been made to Acacia aneura (mulga) is widely distributed in the exploit the N-fi xing characteristic of the genus. Th e Australian arid zone. Its foliage is browsed by ruminants, purpose of this review is to ask why, and to consider especially in times of drought, and its wood is valued for what means might be employed to increase N turning and carving.

17 2. Th e plant

Legumes fi rst appeared on Earth some 70 million Th e Acacia genus includes some of the world’s years ago (Polhill et al. 1981). Th e legumes comprise most beautiful plants. In fl ower, Acacia baileyana¹, three families, viz. Fabaceae, Mimosaceae and A. podalyriifolia and A. pycnantha are fully clad in a Caesalpiniaceae, though some authorities, e.g. Lewis raiment of fl uff y, golden yellow balls that persist for et al. (2001), prefer to regard the legumes as a single several weeks. Indeed, A. pycnantha is Australia’s family (the Leguminosae or Fabaceae) with three fl oral emblem and Wattle Day is celebrated in some subfamilies — Papilionoideae, Mimosoideae and States as the fi rst day of spring. Th e springtime Caesalpinioideae. Within the Mimosaceae, Acacia fragrance of A. mearnsii is a sensuous feature of is much the largest genus (and apparently still the bushland of eastern Australia. Th ere is a grand growing as new species are recognised) — estimated stateliness of form of A. dealbata and A. melanoxylon at some 1200–1300 species by Chappill and Maslin in mature forests. Th e graceful foliage of A. excelsa (1995) and at 1350 species less than nine years and the pensile symmetry of A. pendula create park- later (Turnbull 2004). Nearly 1000 of these occur like settings in the pastoral lands of western New naturally and only in Australia (Maslin and Hnatiuk South Wales. For a traveller in arid country to come 1987; Maslin and McDonald 1996). ‘Th e Flora of upon A. cambagei in full fl ower is an experience to Australia’ lists 955 species of Acacia (Orchard and stir the soul. Low-growing acacias act as shelter, Wilson 2001a,b). Acacia is also widely distributed in sanctuaries, and feeding and breeding grounds Africa (about 144 species), Asia (about 89 species) for small, native mammals and birds. Seedlings and the Americas (about 185 species) (Maslin and make rapid early growth. It is small wonder, then, Stirton 1997; Orchard and Maslin 2003; Turnbull that acacias are sought after as shrubs and trees 2004), but acacias are rare in Europe. Th e genus is for home gardens, parks and roadside verges, and not indigenous to New Zealand and smaller islands to provide visual screens, shade, shelter belts and of the Pacifi c (Greenwood 1978), but is often an wildlife habitats. introduction. Acacias range from herbs (rare) to enormous trees — see e.g. Menninger (1962), but most are shrubs and small trees. Th eir habitats range from arid areas of low or seasonal rainfall to ¹ Th roughout the manuscript, we usually refer to acacias and other plant species by their botanical names. Common moist forests and river banks (Allen and Allen 1981). names and authorities for botanical names are given in the Species are found on all soil types. appendix.

18 nitrogen fixation in acacias utilisation Aesthetics aside, the uses to which Acacia species and acacia products are put are manifold. We will dwell only briefl y and selectively on these. Th e subject has been well reviewed for N-fi xing trees in general by Dommergues et al. (1999) and for acacias in particular by Th omson et al. (1994), Searle (1996), Turnbull et al. (1998a), McDonald et al. (2001) and Maslin and McDonald (2004). Th e genetic resources of useful and potentially useful

acacias have been recorded by Pinyopusarerk (1993). Alison Jeavons Some examples of the wide range of uses of the Acacia pycnantha (golden wattle) is Australia’s fl oral emblem. genus are given in Table 1.

Table 1. Utilisation of (selected) Acacia species Species Uses A. acuminata Charcoal, wood turning A. albidaa Fodderb, soil enrichment A. aneura Fodder, posts, turning, bush foodc A. auriculiformis Environmental rehabilitation, soil stabilisation, fuel wood, posts, pulpwood A. baileyana Cut fl owers/foliage, pollen, gum A. berlandieri Gum A. cambagei Posts, turning, bush food A. catechu Fuel wood A. crassicarpa Tolerance of high water tables A. cyclops Salinity tolerance A. dealbata Pulpwood, gum, cut fl owers/foliage, oils, pollen A. decurrens Pulpwood, timber, fuel wood A. elata Pulpwood A. excelsa Fodder A. gerrardii Fodder A. harpophylla Posts, fuel wood, charcoal A. hebeclada gum (acidic) A. homalophylla Turning

19 nitrogen fixation in acacias

Table 1. (cont’d) Utilisation of (selected) Acacia species Species Uses A. imbricata Cut fl owers/foliage A. implexa Fuel wood, turning, pollen A. irrorata Fuel wood, tannin A. kempeana Fodder A. leucophylla Non-industrial woodd A. mangium Timber, pulpwood A. mearnsii Tannin, oyster poles, mine timber, pollen, fuel wood, charcoal, craft wood, pulpwood, mushroom medium, adhesives, cellulose for rayon, particle board A. melanoxylon Joinery, turning, pulpwood A. mellifera Fodder, honey A. nilotica Non-industrial wood A. notabilis Bush food A. papyrocarpa Bush food, turning A. parramattensis Pulpwood, tannin A. pendula Fodder, turning, fuel wood A. pycnantha Tannin, gum, bush food A. retinodes Salinity tolerance, cut fl owers/foliage, bush food A. salicina Salinity tolerance, soil stabilisation, joinery A. saligna Salinity tolerance, soil stabilisation, tannin, fodder, bush food, (acidic) gum, fuel wood A. senegal Gume A. seyal Non-industrial wood A. silvestris Pulpwood, joinery, fuel wood, posts, pollen, bush food, cut fl owers/foliage, tool handles A. stenophylla salinity tolerance, fodder, posts, fuel wood A. tortilis Fodder, posts, fuel wood, soil stabilisation, charcoal A. trachyphloia Pulpwood A. vernicifl ua Cut fl owers/foliage A. victoriae Fodder, bush food, pollen a More properly Faidherbia albida. b Fodder refers to foliage or fruits grazed or browsed by domestic animals. c Bush food is foodstuff utilised for human consumption through the Australian bush food industry, mainly as fl avourings. d Th e term ‘non-industrial wood’ implies a number of uses including joinery, turning, carving, fencing, charcoal making and fuel wood; it excludes wood suitable for milling as construction timber. e Once widely used, as gum arabic, as adhesive for coating legume seed as an aid to inoculation (Brockwell 1962).

20 nitrogen fixation in acacias

Wood products blackwood, barely does justice to the beauty of its Th e use of acacia wood dates back to ancient times. heartwood which is prized for furniture making, Historians are in general agreement that the joinery and turning. religious icons, the ‘Ark of the Covenant’ and the ‘Altar and Table of the Tabernacle’, were constructed Acacia mangium and A. auriculiformis have been from timber cut from A. seyal and/or A. tortilis widely planted in the tropics of Asia, the former in (Moldenke and Moldenke 1952). plantations for wood-pulp production, the latter mainly by smallholders for non-industrial wood Relatively few species of Acacia grow large enough (Turnbull et al. 1998b). Acacia auriculiformis is for construction timber or furniture making. adapted to infertile soils, including the large areas However, one of these is A. melanoxylon which has a of degraded (Imperata) grasslands in Southeast Asia. wide latitudinal range in Australia (Searle 1996) and Poor stem form has restricted its use, but there is which, in optimum climates, may grow up to 35 m potential for the exploitation of provenances with in height and 1.5 m in diameter (Boland et al. 1984). straight stems (Venkateswarlu et al. 1994) and of its Th e common name of the species, (Tasmanian) hybrids with A. mangium (Kha 1996). David McClenaghanDavid Products from acacia timbers, courtesy of the Bungendore Wood Works Gallery, Bungendore, New South Wales

21 nitrogen fixation in acacias

Many acacias make excellent fuelwood and charcoal (Searle 1995). Fence posts are cut from a number of species (Searle 1996), some of which, e.g. A. cambagei, are resistant to termite attack. Poles of A. mearnsii, cut with the bark intact, are used in oyster farming; the high tannin content of the bark apparently delays degeneration of the poles induced by marine borers (Searle 1996). Other acacia wood products include chips for wood-pulp manufacture of paper, rayon and particle board (Sherry 1971;

Hillis 1996; Mitchell 1998) and for sawdust as a Products Courtesy of CSIRO Forestry and Forest medium for cultivation of edible fungi (Lin 1991). A plantation of Acacia mearnsii (black wattle) near Eldoret Eatel, Kenya. Th e bark of A. mearnsii is a prolifi c source of industrial tannin. Non-wood products

Gums exuded from higher plants, including Humans and animals eat acacias. A conference acacias, are complex carbohydrates (Anderson et in the early 1990s examined the potential of al. 1971, 1984) that are used in food processing acacias as sources of human foodstuff (House and and medicines. Gum arabic, perhaps the most Harwood 1992). Edible acacia seeds have since commercially important of the natural gums, is a been documented (Maslin et al. 1998). Australia’s product of the tree A. senegal, growing mainly in Indigenous people used at least 44 species of Africa (National Academy of Sciences 1979). Acacia for food (Th omson 1992). Th is knowledge is now being utilised in West Africa (Harwood Th e tannins contained in certain wattle barks are 1999; Midgley and Turnbull 2001). To explore the used in water- and weather-proofi ng processes and likelihood that seeds of Australian acacias may prove in the leather industry (Sherry 1971; Yazaki and a useful dietary supplement for human consumption Collins 1997). Th e bark of A. mearnsii is particularly in the Sahel region, the Australian Government in rich in tannins. In Australia, millions of trees were 1997 funded two Aboriginal women from central stripped of their bark to supply tanneries and export Australia to visit an indigenous community in Niger markets between the 1880s and the 1960s (Searle to exchange information (Australian Department 1991). In South Africa, substantial plantations of of Primary Industries and Energy 1997). Th e A. mearnsii grown specifi cally for tannin extraction outcome was productive (cf. Harwood et al. 1999). make that country the world’s major exporter of Leaves of A. pennata subsp. insuavis are used for powdered vegetable tannin (Sherry 1971). culinary and medicinal purposes in Th ailand and India (Bhumibhamon 2002). Wattle seed is now

22 nitrogen fixation in acacias one of the boutique Australian bush foods used as oils extracted from the fl owers of A. dealbata and a fl avouring agent (ANBIC 1996). Grazing animals A. farnesiana are valued as blenders and fi xatives in browse several species of Acacia especially A. saligna. perfume manufacture (Boland 1987). Acacia aneura foliage is used for feeding sheep and cattle in times of drought (Norton 1994). Acacia pollen is a source of protein for honey bees (Boland 1987). In Australia, several species are cultivated for cut fl owers and foliage for both domestic and export markets (Sedgley and Parletta 1993). Fragrant John Brockwell John Acacia peuce (waddy wood) is a rare and endangered species (Leigh et al. 1981). It occurs in small, disjunct populations

John Brockwell John at three general locations, all in inland Australia in very arid Acacia cambagei (gidgee) is a common tree in semi-arid and environments. Th is specimen was photographed in 1999 at arid Australia and may grow to 15 metres. Its wood is hard a reserve dedicated to A. peuce near Birdsville, Queensland. and resistant to termites and is often used for fence posts. Th e mean annual rainfall there is 140 mm.

23 nitrogen fixation in acacias

Many acacias are used for environmental Table 2. Plantings of some Australian species of rehabilitation (Doran and Turnbull 1997). For Acacia in other parts of the world example, they are grown on overburden from mining activities (Langkamp et al. 1979) and Species Approximate Whereabouts of are used to lower watertables beneath saline total area (ha) major plantings soils, including those subject to waterlogging A. crassicarpa 50,000 Sumatra (Biddiscombe et al. 1985; Ansari et al. 1998; Marcar A. mangium 800,000 Indonesia, et al. 1998), in soil stabilisation (Searle 1996; Malaysia Harwood et al. 1998), and to increase productivity A. mearnsii 450,000 South Africa of degraded grassland (Turnbull et al. 1998b). Many A. saligna 500,000 North Africa, Australian acacias are fi re tolerant (Dart and Brown West Asia, Chile 2001), which is a particular advantage for land Source: after Midgley and Turnbull (2001). rehabilitation in wildfi re-prone environments. In fi eld trials, acacias often grow more quickly than other N-fi xing trees. For example, in the fi rst distribution 12 months after outplanting at three locations in In addition to the natural global distribution of Rwanda, the growth of A. mearnsii was superior the genus, acacias are widely grown in plantations. to that of eight other species (Uwamariya 2000). Extensive plantings have been established in Natural hybridisation sometimes occurs between China, India, Indonesia, Malaysia, the Philippines, Acacia species, accompanied by enhancement Th ailand, Vietnam (Turnbull et al. 1998b) and of plant vigour. Kha (2000) reported from South Africa (Sherry 1971). Th e largest plantations Vietnam that the stem volumes of A. mangium/A. are in India where a total area of three million auriculiformis hybrids were about three times hectares (Pandey 1995) has been planted, mainly as great as those of the parents. A factor in the with the spiny species A. catechu, A. leucophloea, improved growth may have been that the root A. nilotica and A. tortilis (Hocking 1993). Th ere nodules of the hybrids were 2–4 times greater are approximately two million hectares planted in number and weight, and perhaps in N-fi xing worldwide (Table 2) with non-spiny Australian capacity, than the nodules of the parental species. acacias, mostly A. mangium, A. mearnsii and A. saligna (Maslin and McDonald 1996; Midgley and plant taxonomy Turnbull 2001). In the past, Australia had few acacia plantations because trees were so readily available in Th e type species of the genus Acacia is A. arabica the wild (Searle 1995). Th at situation now appears (now A. nilotica subsp. nilotica) which is native to to be changing (Mitchell 1998; Neilson et al. 1998; the northern half of the African continent. Th e Byrne et al. 2001). botanical name Acacia is derived from the Greek

24 nitrogen fixation in acacias

‘akis’ meaning point or barb, a reference to the deliberations have been comprehensively dealt with spines. Th e derivation is apt for African species by Orchard and Wilson (2001a,b) in the ‘Flora of which are mainly spiny but not for Australian Australia, Mimosaceae, Acacia’. species which, as a rule, have no spines. Also, the tree once regarded as A. albida² is now Pedley (1986) suggests a plausible scenario for properly known as a Faidherbia (F. albida), a the evolution of Acacia and its subgenera and monospecifi c genus (Vassal 1981) distributed sections. Amongst the legumes, the cosmopolitan, throughout Africa and adjoining regions of West polyphyletic genus Acacia (some 1350 species) Asia. is second only to Astragalus in numerical size. Th e very size of the genus is further complicated A simplistic version of the classifi cation of legumes, by a substantial degree of outcrossing, e.g. with particular reference to Australian acacias, is A. auriculiformis (Khasa et al. 1993), A. crassicarpa illustrated in Figure 1. Of the nearly 1000 species (Moran et al. 1989), A. decurrens (Philp and Sherry of Acacia recognised as Australian, the majority 1946), A. mearnsii (Moff ett 1956) and A. melanoxylon are contained in the subgenus Phyllodineae. Th e (Muona et al. 1991). Moreover, in some species, remainder are accommodated in subgenera Acacia there is a wide range of intra-specifi c diff erentiation (180–190 species) and Aculeiferum (120–130 and diversity, both genetic and morphological, species) (Maslin 2001). e.g. A. acuminata (Byrne et al. 2001), A. aneura (Andrew et al. 2001) and A. tumida (McDonald et Although the rhizobial symbiosis is not a classical al. 2001). Th e diff erentiation within A. aneura is systematic criterion in legume taxonomy, it is now so marked that the species itself is considered a widely recognised that it does have substantial ‘complex’ (Andrew et al. 2001). Taxonomic revision relevance (e.g. Sprent 2001b). Norris (1959) held the of such a genus is probably inevitable. Indeed, a view that the symbiotic characters, nodulation and major revision was proposed nearly 20 years ago N fi xation, expressed during the interaction between (Pedley 1986) but not widely accepted, perhaps African species of Trifolium and strains of Rhizobium because it would be ‘so disruptive’ (Maslin 1995). leguminosarum bv. trifolii (t’Mannetje 1967), Nonetheless, many of Pedley’s (1978) earlier and were useful in the taxonomy of both plants and less-controversial proposals have been adopted for bacteria. He called his concept ‘symbiotaxonomy’. the treatment of acacias in the ‘Flora of Australia’. Th e concept has already found application in the Th is is not to say that revision of some sections and classifi cation of biovars of R. leguminosarum (Kreig individual species is not proceeding. For instance, and Holt 1984). McDonald and Maslin (1998) summarised a ² As a matter of convenience throughout this review, we proposal for a taxonomic revision of A. aulacocarpa use the name A. albida instead of F. albida, although we and its close relatives. Th e outcomes of these recognise that it is obsolete.

25 nitrogen fixation in acacias

A spectacular example of the association between R tropici) and Macroptilium, Macrotyloma and Vigna, symbiotaxonomy and legume taxonomy was the nodulated by slow-growing Bradyrhizobium species. reclassifi cation of the complex genus formerly Symbiotaxonomy apparently also has a potential recognised as Phaseolus (Fabaceae). Legume role in the systematics of Lotus (also Fabaceae) and bacteriologists had long been aware of paradoxes symbiotically related genera (Brockwell et al. 1994). within the complex, viz. diff erent species had fast- and slow-growing root-nodule bacteria and Th ere appears to be some relatedness between there were marked host/bacteria specifi cities in taxonomic classifi cation of the Caesalpiniaceae and nodulation and N fi xation. All this was resolved symbiotic characteristics. Most species within the by Verdcourt (1970a,b). He defi ned several Caesalpiniaceae do not nodulate (Allen and Allen genera within the Phaseolus complex including 1981). However, there are exceptions within the Phaseolus, nodulated by fast-growing species of genus Cassia which contains both nodulating and Rhizobium (now known to include Rhizobium non-nodulating species. When the leguminous tribe leguminosarum bv. phaseoli, R. etli bv. phaseoli, Cassieae subtribe Cassiinae underwent taxonomic R. gallicum bv. gallicum, R. gallicum bv. phaseoli, revision (Irwin and Barnaby 1982; see also Randall R. giardinii bv. giardinii, R. giardinii bv. phaseoli, and Barlow 1998a,b), three separate genera, viz.

Figure 1. A simplistic illustration of taxonomic arrangements within the genus Acacia, with special reference to Australian species — after Tame (1992), derived in part from Pedley (1978). Th is is a practical classifi cation but it does not necessarily refl ect the phylogeny of the genus Acacia.

26 nitrogen fixation in acacias

Cassia, Senna and Chamaecrista, were recognised, several smaller genera. Th e arguments of Pedley and some species, formerly Cassia, were allocated (1986) and Chappill and Maslin (1995), and the to Senna. Two of those were the non-nodulating DNA-based evidence of Miller and Bayer (2001), species, now Senna siamea and S. spectabilis. It is for doing so are compelling. (We have already noted consistent with principles of symbiotaxonomy the reclassifi cation of Acacia albida to Faidherbia that no species within the genus Senna have been albida.) In Australia, debate about how the revision found to nodulate (Allen and Allen 1933; Giller and should be done has led to a polarisation of opinion. Wilson 1991), although the search has not been On the one hand, Pedley (1986) believes inter alia exhaustive. On the other hand, it is inconsistent (i) that the existing type species, A. nilotica, should with those principles that the genus Cassia appears be conserved, with most of the African species still to contain both nodulating and non-nodulating retaining the generic name Acacia, and (ii) that species. the largest existing subgenus Phyllodineae, which contains most of the 1000 Australian species, Until recently, symbiotaxonomic criteria had not should become genus Racosperma. Orchard and been considered within the Mimosaceae. It now Maslin (2003), on the other hand, contend that appears from the work of Harrier et al. (1997) that means should be found to limit the extent of change a taxonomically distinct group of Acacia species that would ensue if the Pedley (1986) proposal native to Africa is unable to form nodules. Th ere were adopted (i.e. new names for more than are also indications (de Faria and de Lima 1998) 1000 species). A somewhat emotional argument, that another group of acacias that occurs naturally specifi cally Australian, against widespread in Central and South America is similarly unable taxonomic change is that the name Acacia is widely to nodulate. Th ese particular species are members recognised here by the general public. It conveys of the subgenus Aculeiferum ser. Americanae. images of trees and shrubs that are familiar in Paradoxically, however, other species within the parks and gardens and in the wild, of a timber that Americanae have been reported as bearing nodules. makes beautiful furniture and turned products, It will be of interest to learn whether taxonomic and of a group of plants with a myriad of functions botanists may, sometime in the future, uncover in everyday life. Th e alternative name Racosperma systematic criteria that separate these non- would not carry the same impact. Turnbull (2004) nodulating groups from nodulating Acacia species at summarises a proposal from Orchard and Maslin the generic level. Further, more specifi c information (2003) that seeks the best of the old and the about non-nodulation in acacias is presented proposed new classifi cations and nomenclature. later, in the section entitled ‘Th e bacteria — acacia Th eir idea is to conserve the generic name Acacia rhizobia in nature’. for the largest subgenus, Phyllodineae. Th is would involve replacing the existing type species, A. nilotica It seems inevitable that the large, complex genus (genus Acacia, subgenus Acacia), with a new type known as Acacia will eventually be revised into species, A. penninervis (genus Acacia, subgenus

27 nitrogen fixation in acacias

Phyllodineae). Th e generic name Acacia would of the perennial nodules of acacia. It seems unlikely then be retained for the largest group, subgenus that they are truly perennial. Perhaps their function Phyllodineae, the species of which predominate in is to provide the host plant with an immediate Australia. Other subgenera would become genera source of atmospheric N as soon as soil moisture Senegalia, Vachellia or Acaciella. All this would mean becomes adequate following a prolonged dry fewer name changes than would be needed if the period. Th is is analogous to the ‘perennial’ nodules Pedley (1986) classifi cation were adopted. A decision of Trifolium ambiguum (Caucasian clover) which is likely in 2005 on how Acacia will be formally overwinter beneath snow, preserve a connection divided. We understand that the impending changes with the vascular system of the roots, and form new will inconvenience some of us but, at the same time, N-fi xing tissue and commence N fi xation at least we appreciate the benefi ts that stem from creating two weeks before the appearance of new roots that out of the old, new genera that are monophyletic can produce new nodules (Bergersen et al. 1963). and of appropriate size (cf. Young 1996). Corby (1971) was amongst the fi rst to record nodule shapes (see also Fig. 2). He later expressed root and nodule morphology the opinion (Corby 1981) that nodule morphology Sprent et al. (1989) comprehensively reviewed might be useful in legume taxonomy. However, his the structure and function of the nodules of idea has never been pursued. woody legumes, including acacias. As a general rule, N-fi xing (eff ective) nodules of Acacia (a) (b) species are of the determinate (elongate) type. Notwithstanding, certain characteristics of acacia nodules are consistent with those of determinate (spherical) types (Lopez-Lara et al. 1993). It is easy to visually identify N-fi xing nodules by the (c) (d) pink, leghaemoglobin-induced colour of their internal tissue. Th ose that are actively fi xing N are usually cylindrical in shape, sometimes coralloid, occasionally multi-lobed. Ineff ective nodules are globose (spherical) and small. Various nodule types are shown in Figure 2. 1 cm

We sometimes observe the occurrence of so-called Figure 2. Classifi cation of the shapes of perennial nodules on acacia. Th eir shape is usually nodules (after Corby 1971) of acacias: elongate with branching (see Fig. 2) and they appear (a) globose, (b) coralloid, (c) elongate to be partly lignifi ed. We know of no investigation with branching, (d) elongate, delicate

28 3. Th e bacteria

acacia rhizobia in nature acacias failing to reveal nodules, e.g. A. glomerosa A painstaking search of the literature, undertaken (Barrios and Gonzales 1971; de Faria and de Lima by Allen and Allen (1981), recorded the occurrence 1998), A. greggii (Martin 1948; Eskew and Ting of nodulation on less than 10% of acacia species. 1978; Zitzer et al. 1996), A. pentagona (Corby Th e fi gure is misleading, however, because it is likely 1974; Harrier et al. 1997), A. polyphylla (de Faria that more than 90% of species simply had not then et al. 1987; de Faria and de Lima 1998) and been examined for the presence of root nodules. A. schweinfurthii (Corby 1974; Harrier et al. 1997). Since 1981, nodulation has been reported for many We could fi nd single, reliable records (Aronson et al. more Acacia species (e.g. Kirkbride 2000; Sprent 1992; Moreira et al. 1992; Odee and Sprent 1992; 2001b), and it now appears probable that all but de Faria et al. 1994; Zitzer et al. 1996; Masutha a few acacias have the capacity to form nodules. et al. 1997) of at least 12 other Acacia species that Nevertheless, there is conclusive evidence that some do not nodulate. It is probably no coincidence species cannot nodulate. that the non-nodulating Acacia species all appear to be closely related as members of the subgenus Th ere are three criteria that, taken together, Aculeiferum, ser. Americanae (de Faria and de Lima constitute good evidence of non-nodulation: (i) 1998). However, two species of the Americanae inability to fi nd nodules on the roots of the legume from Brazil, A. bahaiensis and A. martii, have been at fi eld locations where it is endemic, (ii) failure reported as nodulating plants (Allen and Allen 1981; of the legume to nodulate when it is grown, under Moreira et al. 1992; de Faria et al. 1994). Shaw benign conditions in the glasshouse, in soil taken et al. (1997), reporting on work with nodulating from around roots of the plant in its natural and non-nodulating tree legumes, drew attention habitat, and (iii) no nodulation of the legume in the to the existence of particular root exudates (nod- glasshouse following its inoculation with a large gene-inducing compounds) that played a role in collection, ideally hundreds of strains, of diverse initiating the symbiotic processes that led to nodule rhizobia (J.I. Sprent, pers. comm.). Th e issue of formation in the nodulating species but which were non-nodulation in legumes, as with most negative not present in the non-nodulating trees. Whether data, is contentious. Notwithstanding, there are or not certain acacias fail to nodulate because they a number of reports of careful work on various cannot produce these compounds is unknown.

29 nitrogen fixation in acacias

Th is particular group of acacias (subgenus absent (e.g. Th rall et al. 2001b). Where populations Aculeiferum ser. Americanae) is not endemic to are small (<50 per g), rhizobial inoculation of Australia. Despite unconfi rmed reports to the acacia frequently results in enhanced N fi xation contrary cited by Allen and Allen (1981), we know (Turk et al. 1993). Turk et al. (1993) also report an of no Australian Acacia species (i.e. within the unusual instance in which A. mearnsii responded subgenera Phyllodineae, Acacia and Aculeiferum) to inoculation in the presence of >1000 naturally that does not nodulate. Rhizobia are more common occurring rhizobia per gram of soil, but this fi nding in soil immediately surrounding the root system is inconsistent with experience with other legumes, (rhizosphere soil) than in non-rhizosphere soil e.g. Medicago species (Brockwell et al. 1988) and (Robertson et al. (1995), working with A. senegal). soybean (Glycine max) (Th ies et al. 1991a). It is our own experience with Australian acacias that, even when nodules cannot be found on the While high temperatures, high pH and high roots, rhizobia can be detected in the rhizosphere concentrations of salt limit nodulation and N soil using the bait-plant technique (cf. Date 1980; fi xation by rhizobia/acacia associations (see also Odee et al. 1995). Th ere seems little doubt that the below), there is substantial variability among strains bacteria are almost as widely distributed in nature in their ability to tolerate these conditions (Surange as the genus itself; see, e.g., papers by Miettinen et et al. 1997). We submit that such tolerance is al. (1992) and Amora-Lazcano and Valdes (1992). widespread amongst acacia rhizobia and enables the Provided that the host is present, harshness of organism to survive long periods of environmental the soil environment appears immaterial. Acacia extremes. For instance, it is our observation that, rhizobia occur in arid soils (Barnet and Catt 1991; following rain in very arid parts of Australia, Schulze et al. 1991; Dupuy and Dreyfus 1992), in newly formed roots on Acacia species such as A. dune sands (Barnet et al. 1985; Hatimi 1995), in tetragonophylla quickly become nodulated, indicating surface soils and at depth (Dupuy et al. 1994), and a presence of rhizobia. Th e ensuing N fi xation would sometimes at great depth — 34 metres (Dupuy and provide the plant with a supply of atmospheric N Dreyfus 1992). and probably some ecological advantage. However, in such environments, the N supply is likely to be In the fi eld, the size of naturally occurring short-lived since N fi xation will cease as soon as populations of acacia rhizobia varies considerably. soil moisture stress becomes severe — e.g. Sprent Numbers as high as 2.3 × 10⁵ per gram of soil have (1971a,b); see also the later section dealing with been recorded (e.g. Odee et al. 1995). In many other soil moisture as an environmental factor limiting N situations, however, numbers may be very low or fi xation in the fi eld.

30 nitrogen fixation in acacias taxonomy of acacia rhizobia to be Rhizobium. It was not until Jordan (1982) Th e root-nodule bacteria (rhizobia³) that nodulate that the new genus Bradyrhizobium was created to and fi x N with legumes belong to at least six genera accommodate the slow growers. Bradyrhizobium within the Rhizobiaceae: Rhizobium, Bradyrhizobium, japonicum, which nodulates soybean, is the type Sinorhizobium, Mesorhizobium, Azorhizobium species of Bradyrhizobium and one of relatively and Allorhizobium. Th ese genera belong to three few named species in the genus. Th ere are other distinct phylogenetic branches within the α-2 groups of strains that apparently belong to subclass of Proteobacteria. Quite recently, new Bradyrhizobium but which have not been assigned genera of bacteria that nodulate legumes have to a species. It is customary to describe such strains been described. Sy et al. (2001) discovered a strain as ‘Bradyrhizobium sp.’ followed, in parenthesis, by of a Methylobacterium sp. in nodules of Crotalaria the name of the host genus, thus: Bradyrhizobium spp. that constituted a fourth branch within the sp. (Lupinus). We accept this procedure for unnamed α-2 subclass. Jaftha et al. (2002) characterised, as slow-growing strains of acacia rhizobia that Methylobacterium, the pink bacteria that nodulate belong to Bradyrhizobium, thus Bradyrhizobium sp. Lotononis bainesii. At much the same time, there (Acacia), and for unnamed fast-growing strains were reports of species of Burkholderia, belonging to of acacia rhizobia that belong to Rhizobium, thus the β-subclass of the Proteobacteria, isolated from Rhizobium sp. (Acacia). Other slow-growing acacia legume root-nodules. Moulin et al. (2001) reported rhizobia include Sinorhizobium saheli; other fast- the identifi cation of a Burkholderia from nodules growing strains include Mesorhizobium plurifarium. of Aspalathus carnosa, and Vandamme et al. (2002) Some strains of rhizobia that nodulate acacia may noted that B. tuberum and B. phymatum nodulated belong to still other as yet unnamed genera of the roots of tropical legumes. More recently, Ngom the Rhizobiaceae and other families of bacteria. et al. (2004) isolated bacteria of the Ochrobactrum Barnet et al. (1985), for instance, recognised clade from the root-nodules of A. mangium. extra-slow-growing rhizobia that formed nodules on acacia. Also, Yonga (1996) implicated a new Fred et al. (1932) distinguished two groups of genus in nodulating acacias. She called it ‘Pseudo- root-nodule bacteria, the basic distinction being Bradyrhizobium’. Th e taxonomy of the Rhizobiaceae rate of growth: fast growers that acidify culture as a whole is dealt with comprehensively by medium, and slow growers that do not acidify Young (1996), Young and Haukka (1996), Young medium and tend to be associated with tropical et al. (2001) and Sawada et al. (2003), and neatly legumes (Norris 1965). Both groups were considered summarised by Sprent (2001b). Th e system of classifi cation as defi ned by these authors, plus more ³ In this review, we use the terms ‘rhizobia’, ‘root-nodule recent additions, is shown in Table 3. bacteria’ and ‘acacia rhizobia’ interchangeably to refer collectively to bacterial strains of genera of accepted legume symbionts (Rhizobium, Bradyrhizobium, Sinorhizobium, (e.g. Methylobacterium, Burkholderia and Ochrobactrum), Mesorhizobium, Azorhizobium and Allorhizobium), to strains and to strains of other genera yet to be named and properly of other genera not yet widely accepted as symbionts classifi ed — e.g. ‘Pseudo-Bradyrhizobium’ (Yonga 1996).

31 nitrogen fixation in acacias

Table 3. Nomenclature of rhizobia Genus Specifi c name Hosts (not necessarily exclusive) Additional references Rhizobium R. leguminosarum Frank (1889) bv. trifolii Species of Trifolium bv. viciae Species of Pisum, Lathyrus, Lens and Vicia bv. phaseoli Species of Phaseolus R. etli Segovia et al. (1993); Wang et al. (1999b) bv. mimosae Mimosa affi nis bv. phaseoli Phaseolus vulgaris R. gallicum Amarger et al. (1997) bv. gallicum P. vulgaris bv. phaseoli P. vulgaris R. giardinii Amarger et al. (1997) bv. giardinii P. vulgaris bv. phaseoli P. vulgaris R. galegae Lindstrom (1989) bv. giardinii Galega offi cinalis bv. phaseoli G. orientalis R. hiananense Desmodium sinuatum cited from Sprent (2001b) R. huautlense Sesbania herbacea Wang et al. (1998) R. mongolense Medicago ruthenica Van Berkum et al. (1998) R. tropici Phaseolus vulgaris, Leucaena esculenta, Martinez-Romero et al. (1991) L. leucocephala R. yanglingense Amphicarpaea trisperma, Coronilla varia, Tan et al. (2001) Gueldenstaedtia multifl ora Bradyrhizobium B. japonicum Glycine max Jordan (1982) B. elkanii G. max Kuykendall et al. (1992) B. liaoningense G. max Xu et al. (1995) B. yuanmingense Species of Lespedeza Yao et al. (2002) B. betae Unknown B. Lafay and J.J. Burdon, unpublished data B. canariense Unknown B. Lafay and J.J. Burdon, unpublished data Several unnamed Genera of many species that are species of nodulated by slow-growing strains of Bradyrhizobium rhizobia

32 nitrogen fixation in acacias

Table 3. (cont’d) Nomenclature of rhizobiaa Genus Specifi c name Hosts (not necessarily exclusive) Additional references Sinorhizobiuma S. abri Abrus precatorius Ogasawara et al. (2003) S. indiaense Sesbania rostrata Ogasawara et al. (2003) S. meliloti Species of Medicago, Melilotus and Dangeard (1926) Trigonella S. medicae Species of Medicago Rome et al. (1996a,1996b) S. adhaerens Not known Willems et al. (2003); Young (2003) S. arboris Acacia senegal Nick et al. (1999) S. fredii Glycine max, Cajanus cajan and Vigna Scholla and Elkan (1984) unguiculata S. kostiense Acacia senegal Nick et al. (1999) S. kummerowiae Not known cited from Young (2003) S. morelense Leucaena leucacephalab Wang et al. (2002) S. saheli Species of Acacia and ‘a number of other de Lajudie et al. (1994) tree genera’ S. terangae de Lajudie et al. (1994) bv. acaciae Species of Acacia bv. sesbaniae Species of Sesbania S. xinjiangense Glycine max Peng et al. (2002) Mesorhizobium M. loti Species of Lotus, Anthyllis and Lupinus Jarvis et al. (1982) M. amorphae Amorpha fruticosa Wang et al.. (1999a) M. ciceri Cicer arietinum Nour et al. (1994) M. huakuii Astragalus sinicus Chen et al. (1991) M. mediterraneum Cicer arietinum Nour et al. (1995) M. plurifarium Species of Acacia, Chamaecrista, Leucaena de Lajudie et al. (1998b) and Prosopis M. septentrionale Astragalus adsurgens Gao et al. (2004) M. temperatum A. adsurgens Gao et al. (2004) M. tianshanense Various legumes including Glycine max Chen et al. (1995) Azorhizobium A. caulinodans Nodulates stems and roots of Sesbania Dreyfus et al. (1988) rostrata Allorhizobium A. undicola Neptunia natans de Lajudie et al. (1998a) Blastobacter B. denitrifi cans Aeschynomene indica Van Berkum and Eardly (2002) Burkholderia Burkholderia sp. Aspalathus carnosa Moulin et al. (2001)

33 nitrogen fixation in acacias

Table 3. (cont’d) Nomenclature of rhizobiaa Genus Specifi c name Hosts (not necessarily exclusive) Additional references B. caribensis ‘Tropical legume(s)’ Vandamme et al. (2002) B. phymatum ‘Tropical legume(s)’ Vandamme et al. (2002) B. tuberum ‘Tropical legume(s)’ Vandamme et al. (2002) Devosia D. neptuniic Neptunia natans Rivas et al. (2002) Methylobacterium Methylobacterium Lotononis bainesii Jaftha et al. (2002) sp. M. nodulans Species of Crotalaria Sy et al. (2001); Jourand et al. (2004) Ochrobactrum Ochrobactrum sp. Acacia mangium Ngom et al. (2004) Ralstoniaa R. taiwanensis Mimosa spp. Chen et al. (2001, 2003) Source: earlier references from generally after Young (1996), Nick (1998), Van Berkum and Eardly (1998) and Sprent (2001b). a Th e generic name Ensifer (Young 2003; cf. Willems et al. 2003) may have nomenclatural priority over Sinorhizobium. Th e generic names Wautersia (Vaneechoutte et al. 2004) and Cupriavidus (Vandamme and Coenye 2004) have been proposed as alternative nomenclature for Ralstonia. At the time of updating this table (February 2005), these alternative names have not been generally accepted. b Although the novel species Sinorhizobium morelense was isolated from nodules of Leucaena leucocephala, it did not form nodules when re- inoculated on to the host plant (Wang et al. 2002); however, a strain closely related to the novel strain was able to nodulate L. leucocephala. c Th e specifi c name ‘neptunii’ is tentative. diversity of acacia rhizobia strains described as typically Rhizobium, and from Lange (1961) was one of the fi rst to recognise great rain forest and coastal heathlands, other strains diversity amongst rhizobia from native legumes described as typically Bradyrhizobium. A third type, growing in south-western Australia. Th e extent of strains from alpine areas, was extra-slow-growing that diversity has been confi rmed by Marsudi et al. and was thought to represent another genus. Later, (1999) with the rhizobia isolated from Acacia saligna Barnet et al. (1985) isolated this third type from A. growing in the same general area. Lafay and Burdon suaveolens and A. terminalis growing on coastal dune (1998), using a molecular approach, identifi ed sand. At the time, Pedley (1987) also considered that similar diversity in the structure of rhizobial three genera were responsible for acacia nodulation. communities nodulating acacias growing in forests Similar observations have been reported for the in south-eastern Australia. rhizobia of African acacias (Habish and Khairi 1970; Dreyfus and Dommergues 1981). Indeed, Dreyfus Lawrie (1981, 1985) demonstrated that nodulation and Dommergues (1981) isolated both Rhizobium of Australian acacias was induced by both Rhizobium and Bradyrhizobium from nodules on the same tree. and Bradyrhizobium. Barnet and Catt (1991) Even more extraordinary was the isolation of both investigated Acacia rhizobia from diverse localities in genera from the same nodule taken from a root of A. New South Wales. Th ey obtained, from the arid zone, abyssinica (Assefa and Kleiner 1998).

34 nitrogen fixation in acacias

Characterisation of naturally occurring populations Prosopis chilensis were extremely diverse in of acacia rhizobia, using morphological, biochemical, physiological and biochemical features, as well as symbiotic, electrophoretic, chromatographic in cross-nodulation patterns. Haukka et al. (1996) and molecular characters, has been reported by used molecular technology to assess the diversity of a number of investigators including Zhang et al. rhizobia isolated from the nodules of A. senegal and (1991), Amora-Lazcano and Valdes (1992), de P. chilensis. Sequence comparison indicated that one Lajudie et al. (1994), Dupuy et al. (1994), Lortet et strain was closely similar to Rhizobium haukuii and al. (1996), Haukka et al. (1996, 1998), Milnitsky that the others belonged to the genus Sinorhizobium. et al. (1997), Nuswantara et al. (1997), Odee et Similar degrees of diversity, found using diff erent al. (1997), Swelin et al. (1997), Zahran (1997), methods, were recorded by Amora-Lazcano and Khbaya et al. (1998), Vinuesa et al. (1998) and Valdes (1992) and Haukka and Lindstrom (1994). Marsudi et al. (1999). Frequently, these data have Taxonomic positions of rhizobia from A. albida were been employed to defi ne relatedness among strains determined by Dupuy et al. (1994) with sodium isolated from mixed soil populations, using various dodecyl sulphate–polyacrylamide gel electrophoresis forms of pattern analysis. It is usual in these studies (SDS–PAGE). Most strains belonged to eight clusters to distinguish several major clusters or groups which contained representatives of Bradyrhizobium (of identity). Th e ratio of the number of clusters to japonicum, B. elkanii and Bradyrhizobium sp. Th e the total number of strains examined appears to same rhizobia were also characterised with the fall in the range 1:10 to 1:20. In addition, there is Biolog™ system. (Biolog™ = sole carbon source invariably a number of individual strains that are utilisation.) Th e results obtained from the two unrelated to any of the others. At the strain level, procedures were poorly correlated. such procedures are often used for individual strain identifi cation, which is an essential tool for studying It is obvious that acacia rhizobia are diverse rhizobial ecology, inter-strain competitiveness and organisms. It is also clear that acacia species the success of inoculation. belonging to the same taxonomic section, individual species and sometimes the same tree often form Th e same procedures are also used at higher levels nodules, and perhaps fi x N, with bacteria from of taxonomic classifi cation, viz. biovar, species diverse taxonomic groups. Orderly classifi cation of and genus. For example, results from a thin- the rhizobia has been diffi cult, but recent procedural layer chromatography analysis of the nod factors modifi cations are now leading to groupings of synthesised by rhizobia from Acacia and Sesbania led strains that are reproducible using diff erent Lortet et al. (1996) to propose that the two groups methods. For example, McInroy et al. (1998), be named, respectively, Sinorhizobium teranga bv. working with 12 rhizobial isolates from African acaciae and S. teranga bv. sesbaniae. Zhang et al. acacias and other tropical woody legumes, reported (1991), using numerical analysis of 115 characters, for the fi rst time congruence between the results of concluded that the rhizobia of A. senegal and Biolog™ analysis and genotypic fi ngerprinting.

35 nitrogen fixation in acacias comment on the taxonomy and root infection and nodule diversity of acacia rhizobia formation Many investigations of the diversity of rhizobia As with many other legumes, most infection of contribute little towards a better understanding acacias by root-nodule bacteria appears to take place of the complexities of rhizobial taxonomy except via root hairs — e.g. A. albida (Gassama-Dia 1997); to confi rm what is already very well known, viz. A. senegal (Rasanen and Lindstrom 1999) — even that the fi eld is very complex. One-off papers that though acacia root hairs are often sparse (Rasanen use a small number of taxonomic tools to deal et al. 2001). However, alternative routes of infection with a small collection of strains assembled from of some species, viz. wound (crack) infection — e.g. a small number of plants growing in a relatively Allen and Allen (1940) and Chandler (1978) — and small area are particularly unhelpful. We believe infection through intact roots — e.g. Dart (1977) that the best prospect for elucidating the fi eld lies and de Faria et al. (1988) — cannot be ruled out. in ongoing programs using a polyphasic approach Gassama-Dia (1997) noted that nodulation occurred and pooling the resources of several laboratories. promptly following inoculation of young seedlings. Progress has been made where this has been done. Rasanen and Lindstrom (1999) reported that the For example, a group of strains of acacia rhizobia infection process was normal at relatively high root was assigned to ‘gel electrophoretic cluster U’ by temperatures below 38°C but that nodule formation de Lajudie et al. (1994). Characterisation of the was retarded at 38° and 40°C and ceased altogether group by electrophoresis of total cell protein, at 42°C. auxanographaphic tests, DNA base composition, DNA-DNA hybridisation, 16S rRNA gene However, some Acacia species — e.g. A fl eckii, sequencing, repetitive extragenic palindromic A. macrostachya (Harrier 1995) — appear not to PCR, and nodulation tests gave de Lajudie and 11 produce root hairs. Rhizobia probably enter such co-workers (1998b) suffi cient confi dence to propose plants by infecting breaks in the root system formed former cluster U as a new species, Mesorhizobium by emergence of lateral roots. Th is mode of infection plurifarium, and to deposit a type strain in the LMG was reported fi rst by Allen and Allen (1940) in Arachis (Laboratorium voor Microbiologie, Universiteit hypogea (peanut). Some strains of rhizobia may enter Gent) collection of bacterial strains (see also their diff erent hosts either by root-hair infection or Table 3). by break infection (Sen and Weaver 1984). Likewise, certain host legumes — e.g. white clover (Trifolium It is still fair to say, as did Young (1996), that the repens) (Mathesius et al. 2000) — may utilise both taxonomy of the Rhizobiaceae is ‘in a state of fl ux’. means of infection. Th e subject of legume root However, continuing rapid advances in the fi eld (e.g. infection leading to nodulation has twice been Sawada et al. 2003) make it seem likely that before comprehensively reviewed by Sprent (1994b, 2001b). long it will be possible to be more confi dent about the classifi cation of acacia rhizobia.

36 nitrogen fixation in acacias

Acacia species have distinctly fi brous root systems, Strains of non-tumour-forming Agrobacterium particularly in the seedling stage. We know of no lacking genes for N fi xation have been isolated published results of measurements of the frequency from nodules of several African legumes, including on acacia roots of sites (foci) that are available for Acacia species (de Lajudie et al. 1999). Th e precise infection and subsequent nodulation by rhizobia. role, if any, of these organisms in the host/rhizobial However, our own unpublished observations symbiosis is not understood. It is possible that indicate that infection foci occur at high frequency. the Agrobacterium may sometimes act as a vector For example, we have noted in acacia forests that to assist the rhizobia in the early stages of root the fi brous mass of surface roots immediately infection. Th ere is no evidence for this proposition beneath the layer of leaf litter is often nodulated in except that a comparable phenomenon has been great abundance. Under less favourable conditions postulated for the infection of pea (Pisum sativum) for nodule formation, Hogberg and Wester (1998) by R. leguminosarum bv. viciae (Van Rensburg and observed a substantial reduction in the fi ne root Strijdom 1972a,b). biomass of acacias planted on tractor tracks left behind as a consequence of logging. Th is was rhizobial products accompanied by reductions in both root nodulation and mycorrhizal infection. Th ere is little published work on those bacterial products of acacia rhizobia that might infl uence various aspects of the symbiotic system. Th e mechanisms are probably similar whatever the rhizobial species. For instance, Bhattacharyya and Basu (1992) showed that Bradyrhizobium isolated from A. auriculiformis induced the production of the auxin, indole acetic acid (IAA), from tryptophan in culture. Likewise, Keff ord et al. (1960) detected tryptophan in the root medium of axenic cultures of Trifolium subterraneum (subterranean clover). Th e trytophan was partially converted to IAA

Ben Bohlool when the cultures were subsequently inoculated A rhizobial infection thread in a root hair of Trifolium repens with R. leguminosarum bv. trifolii. It has long been (white clover). An identical phenomenon, an early stage in considered that auxin has a role in the formation and the processes leading to nodule formation, occurs in Acacia growth of legume nodules (Th imann 1936, 1939). species. In acacias, infection threads often initiate from sac- like structures that themselves develop from the point of Extracellular polysaccharides are natural products of primary infection of the root hair by the rhizobia (Rasanen the growth of rhizobia (e.g. Dudman 1976) including et al. 2001). those in isolates from the nodules of A. cyanophylla

37 nitrogen fixation in acacias

(Lopez-Lara et al. 1993, 1995) and A. senegal competitiveness. It is known that the capacity of (Lindstrom and Zahran 1993). When some strains, siderophores to sequester and bind iron molecules including acacia rhizobia (J. Brockwell, unpublished inhibits iron-dependent fungi that may otherwise data), are grown in culture medium, polysaccharide parasitise or compete with the bacteria. Siderophore production may be copious. An involvement has production has been recorded for the rhizobia of been postulated (Dudman 1977) for polysaccharides A. mangium (Lesueur et al. 1993; Lesueur and Diem in strain specifi city, i.e. the ability of certain 1997). strains to infect some legumes but not others. Polysaccharide-based encapsulation of rhizobial Gene products of rhizobial cells are the catalysts of cells (Dudman 1968) may be a mechanism for the the intimate processes involved in the regulation survival of the bacteria when they are exposed to of nodule formation and nitrogen fi xation. environental stress while free-living in the soil. Understanding in this area has advanced rapidly (Vincent 1980; Kennedy et al. 1981; Caetono- Siderophore production by rhizobia may also Annolles and Gresshoff 1991; Dakora et al. 1993; be a survival mechanism and/or an aid to Bladergroen and Spaink 1998). Alison Jeavons Acacia decurrens

38 4. Th e symbiosis

Th e ancestors of rhizobia may have been on Earth Th e benefi ts to the micro-organism are pronounced. many millions of years before the legumes appeared Th ies et al. (1995) showed that populations of (Sprent 2001a). While how and when the symbiosis Bradyrhizobium in soil were substantially enriched as between legumes and root-nodule bacteria evolved a result of cropping with a homologous legume. Th at remains a mystery, it is a subject that has aroused is, growth of the legume stimulated multiplication much speculation (e.g. Raven and Sprent 1989; of the rhizobia with which it formed nodules. In Young and Johnston 1989; Herendeen et al. 1992; elegant experiments with Glycine max (soybean) Young 1993; Sprent 1994a; Soltis et al. 1995) and and Bradyrhizobium japonicum, Reyes and Schmidt some controversy (e.g. Norris 1956, 1958, 1965; (1979) and Kuykendall et al. (1982) demonstrated Parker 1957, 1968). that the majority of the population of rhizobia in the soil and/or in nodules formed on soybean roots What is not in question is that legumes and were derived from organisms that had occupied rhizobia are not dependent on each other for their nodules on the roots of the previous year’s soybean very existence. Th ere are numerous authenticated crop. instances of legumes that, like non-symbiotic plants, successfully complete their life cycles, Th e advantage of the symbiosis to the host is less including reproduction, without ever becoming pronounced. Nevertheless, the ability to access a nodulated or fi xing N. Indeed, many species of the source of N unavailable to non-symbiotic plants Caesalpiniaceae (Brockwell 1994; Sprent 1995, helps the legume to compete ecologically as a 2001a) and some members of the other legume volunteer or a weed, or to produce agronomically as families — e.g. the genus Chaetocalyx (Fabaceae) a crop or pasture plant. Because it is less dependent (Diatloff and Diatloff 1977) — do not nodulate at than the organism, the legume can perhaps be all. Likewise, there are records of the root-nodule considered as the major partner of the symbiosis. organism surviving for long periods in the fi eld in the absence of a host that it can nodulate (e.g. Humans have long been aware of the benefi ts Bergersen 1970) or in dry soil in storage. As a rule, of symbiotic N fi xation, even if they did not however, the symbiosis confers advantages on both understand the process. In the 12th century BC, partners. for example, Th eophrastus, a Greek philosopher, wrote about the re-invigorating eff ect of growing

39 nitrogen fixation in acacias legumes on exhausted soil — cited by Fred et al. suitable for abundant symbiotic N fi xation (e.g. (1932). Th e global amount of biological N fi xation Morley and Katznelson 1965). Th e result, which is a matter for conjecture. Estimates by Burns and is an example of what we mean when we speak of Hardy (1975) and Paul (1988), augmented by Bunt harnessing N fi xation, was the establishment of (1988), suggest that the annual total approaches 38 million hectares of pasture, containing at least or exceeds 200 million tonnes. Much of this N is 5% subterranean clover (Pearson et al. 1997), on derived from terrestrial natural systems, agriculture previously N-defi cient land. and forestry. According to calculations made by Peoples et al. (1995a), symbiotic systems in arable One of the fi rst leguminous trees to be utilised at land and permanent pasture account for 80–84 least partly for its N-fi xing ability was leucaena million tonnes. Nitrogen fi xation by leguminous (Leucaena leucocephala). Leucaena has a long history trees in forests — and by symbiotic non-leguminous as a shade tree for coff ee (Coff ea spp.) and cocoa trees, e.g. the Casuarina (Allocasuarina)/Frankia (Th eobroma cacao). Its forage value was fi rst noted association — would add to the estimate. Indeed the in Hawaii by Takahashi and Ripperton (1949) who fi gure may be higher still, since the Peoples et al. reported annual dry matter production of 20–25 (1995a) calculations probably underestimated the tonnes per hectare, and foliage N amounting to substantial quantity of fi xed N partitioned in plant 400–600 kg per hectare. A direct outcome of this roots (Zebarth et al. 1991; McNeill et al. 1997; Khan observation was the utilisation of leucaena as the et al. 2000; Unkovich and Pate 2000; Peoples and leguminous component of pastures sown to provide Baldock 2001). grazing and browse for beef cattle in northern Australia (Griffi th Davies and Hutton 1970) and Whatever the fi gure for global biological N elsewhere in the world (Vietmeyer 1978). fi xation, probably about half is due to N fi xation by legumes. A substantial component of that is With the exception of some green manure plants contributed by agricultural legumes particularly such as Chinese milk vetch (Astragalus sinicus) and where plant-nutrient defi ciencies in the soil, other a few other herbaceous species noted by Giller and than N, have been corrected. A striking instance Wilson (1991), legumes are only rarely cultivated is the exploitation of the exotic annual self- merely for their capacity to fi x N. Nitrogen fi xation regenerating legume, subterranean clover (Trifolium is a secondary consideration. Clover is primarily subterraneum), for the benefi t of Australian pastoral a forage, leucaena is grown as a browse or a shade enterprises and as the legume component of ley- tree, other species yield food and fi bre, and so farming systems (Puckridge and French 1983). on. Acacia species have manifold uses but their Application of phosphorus, and sometimes minor paramount product is wood. Th is is attested to, for elements, accompanied by sowing subterranean instance, in the proceedings of an international clover inoculated with eff ective Rhizobium conference held in Vietnam in 1997 (Turnbull et leguminosarum bv. trifolii, provided conditions al. 1998a) dealing with plantings of Australian

40 nitrogen fixation in acacias acacias in various parts of the world. None of the 60 Th e acetylene reduction assay (ARA) contributions to the workshop proposed that Acacia Th e application of ARA for measuring N fi xation in spp. be grown solely as a source of biological N. It is nodules of Acacia spp. is detailed by Hansen et al. a bonus that a wood producer should also fi x N. In (1987). ARA is a widely used diagnostic tool dating this review, we show how that attribute might be back to Dilworth (1966) and Hardy and Knight exploited without aff ecting wood production. (1967). It is apt for measuring nitrogenase activity (N fi xation) at an instant in time, particularly that of free-living diazotrophs growing in culture medium. measurement of nitrogen Its application, especially to higher plants including fixation legumes, however, is fraught with many pitfalls, as Eff ective management of biological N fi xation listed by Sprent (1969), Witty (1979), Van Berkum ultimately relies upon a capacity to measure and Bohlool (1980), Van Berkum (1984), Boddey it accurately (Peoples and Herridge 2000). (1987), Giller (1987), Sloger and Van Berkum (1988) Descriptions and/or appraisals of the various and Witty and Minchin (1988). One of these, an methods for measuring N fi xation are given in acetylene-induced decline in nitrogenase activity Chalk (1985), Shearer and Kohl (1986), Ledgard during assay, is demonstrated by Sun et al. (1992a) and Peoples (1988), Peoples and Herridge (1990), using ARA for estimating the nitrogenase activity Danso et al. (1993), Herridge and Danso (1995) and in nodulated roots of A. mangium. We believe Unkovich et al. (1997). that ARA is generally unsuitable for quantifying N fi xation (nitrogenase activity) in acacias and Th ere are four principal methods: (i) the acetylene other leguminous trees, but is a useful qualitative reduction assay (ARA) as a measure of nitrogenase measurement. Notwithstanding, we have cited a activity which, in turn, is an index of N fi xation; number of investigations that used ARA to quantify (ii) the xylem-solute method, which measures N fi xation in the glasshouse, nursery and fi eld. N-containing compounds that are products of N fi xation and are carried from the root nodules Th e xylem-solute method to the shoots in xylem sap; (iii) the N-diff erence method, which measures the diff erence in N uptake Many tropical legumes transport most of the between a N-fi xing legume and a non-N-fi xing nitrogenous products of their N fi xation as control plant; and (iv) ¹⁵N-isotopic methods, which ureides. Th e greater the dependence of a plant on measure proportions of ¹⁵N/¹⁴N in N-fi xing legumes fi xed N, the higher the proportion of ureides to and non-N-fi xing controls. Th ere are two popular nitrates plus amino compounds in the xylem sap. applications of ¹⁵N-isotopic techniques: (a) involving Th is characteristic can be exploited to assay the the use of artifi cial ¹⁵N enrichment of the soil, and proportion of ureides in the nitrogenous compounds (b) involving the use of natural enrichment (natural in bleeding or vacuum-extracted xylem sap and abundance) of ¹⁵N in the soil. for constructing calibration curves for estimating

41 nitrogen fixation in acacias

N fi xation (McClure and Israel 1979; Herridge accurate and reliable, but is less so when used in the 1982). A recent paper (Herridge and Peoples 2002) fi eld. Th e diffi culty lies in the selection of the control suggests that quite accurate estimates of legume so that both N-fi xing and non-N-fi xing plants N fi xation can be obtained by the ureide assay of contain the same amounts of soil-derived N in their a single sample of xylem sap. Th e fi xed N of most shoots. Diff erences between the two plant types other symbiotic legumes is transported from the in their capacities to extract and accumulate soil N nodules as amides. Assays have been similarly almost invariably exist. Even when a non-nodulating developed to measure the proportion of amides to isoline of the test legume is used as a control, fi eld other nitrogenous compounds in the xylem sap of results may be unreliable (Boddey et al. 1984). such legumes (Peoples et al. 1986, 1987). Th e amide assay is less sensitive than the ureide assay. Th e ¹⁵N isotopic methods

Xylem sap extraction is more easily achieved Th ese methods separate legume N into two with herbs than with woody species. Besides, a fractions: (i) N originating from soil N, and (ii) N lethal sampling, which is more acceptable for crop originating from atmospheric N. plants than for trees, is needed for most effi cient extraction of xylem sap. Acacias appear to export ¹⁵N enrichment the nitrogenous products of their N fi xation as two amides, asparagine and glutamine. Hansen Almost all soils are naturally enriched with ¹⁵N and Pate (1987b) suggest that the amide (in xylem compared with the ratio of ¹⁵N/¹⁴N in atmospheric sap) technique is not satisfactory for estimating N. Th e level of natural enrichment of plant- N fi xation in the Acacia spp. found in Western available N in soil can be increased artifi cially by Australian forests. If that is so, the same constraint incorporation of a ¹⁵N-enriched nitrogenous salt. is likely to apply everywhere. Th e use of methods involving soil augmentation with ¹⁵N to estimate N fi xation has been comprehensively reviewed (e.g. Chalk 1985; Danso Th e nitrogen diff erence method 1988). Provided that the N-fi xing test plant and Th is is the simplest method. It is based on the its non-N-fi xing control are well matched, the principle that the diff erence in N uptake between an technique gives a reliable estimate of the proportion inoculated, nodulated legume and an uninoculated of legume N derived from atmospheric N (%Ndfa), control represents the amount of N fi xed. When which is averaged over time. A major disadvantage applied under bacteriologically controlled conditions is that applied N, particularly nitrate, may interfere using N-free media or substrate in the laboratory with nodulation (cf. Tanner and Anderson or glasshouse (Brockwell et al. 1982), the method is 1964). Th e technique requires sophisticated instrumentation.

42 nitrogen fixation in acacias

Natural ¹⁵N abundance Chalk and Ladha (1999) are critical of both Th e natural ¹⁵N abundance method depends on ¹⁵N-enriched and ¹⁵N natural abundance isotope natural enrichment of plant-available N in the soil dilution methods, because of the non-uniform with ¹⁵N to provide the benchmark diff erence in distribution of isotopic N through the soil profi le ¹⁵N/¹⁴N ratios between atmospheric N and soil whether the discrimination is natural or imposed by N. Otherwise, the considerations are similar to ¹⁵N enrichment. Th e authors are suspicious of the those for ¹⁵N enrichment techniques, except that reliability of reference plants used to benchmark certain limitations diff er (Mariotti et al. 1983; the extent and variability of isotopic discrimination Shearer and Kohl 1986). Th e major advantage of in the soil. Th e consequences of their somewhat the natural abundance technique is that, because gloomy appraisal can be moderated by selection of no pre-treatment with ¹⁵N salt is required, it can be non-N-fi xing reference plants with root geography applied to existing experiments or to trees growing as similar as possible to that of the N-fi xing target in plantations or forests. plant.

Both of the ¹⁵N isotopic methods are likely to Th ere are, of course, in studies with shrubs and underestimate total N fi xation in legumes because trees, other diffi culties that limit the accuracy they take no account of fi xed N in underground of estimating N fi xation (Boddey et al. 2000a). plant parts. Recent research fi ndings for pasture Th ey include perennial growth, seasonal and legumes (Zebarth et al. 1991; McNeill et al. 1997; yearly variations in N assimilation (e.g. Ladha et Khan et al. 2000; Unkovich and Pate 2000; Peoples al. 1993; Peoples et al. 1996), and large plant-to- and Baldock 2001) indicate that 50% or more of the plant diff erences in growth and nodulation which total N may be partitioned below-ground. can occur within species and even within a single provenance (e.g Burdon et al. 1999). Paparcikova et al. (2000) noted a further complication: in the consideration of procedures for Amazon jungle, there was little if any N fi xation measuring n fixation by the leguminous component of primary forest We are indebted to Peoples et al. (1989) from whom whereas, following clearing, those same tree we have summarised procedures for measuring N legumes fi xed N in secondary vegetation sites. fi xation. Th eir monograph describes in substantial detail the various methods we have listed, with Conscious of the aforementioned constraints, emphasis on application to fi eld-grown legumes. we recommend two procedures for assessing N Th ey pay particular attention to the proper fi xation in acacias. When measuring the symbiotic evaluation and interpretation of analytical data, to eff ectiveness of strains of rhizobia, or the response applications, to advantages and to limitations. In of acacia seedlings to rhizobial inoculation, or using comparing the methods, they stress that there is no the ‘whole-soil’ inoculation technique (Bonish 1979; ‘correct way’ to measure N fi xation. Brockwell et al. 1988) to estimate the N-fi xing

43 nitrogen fixation in acacias capacity of a mixed population of rhizobia in attention to the many pitfalls that might be soil, it is feasible to work with N-free media and encountered when using the technique, they suggest bacteriological control in tubes (Th ornton 1930), that, used prudently, it currently represents the best pouches (Somasegaran and Hoben 1994), Leonard means of measuring symbiotic N fi xation by woody jars (Leonard 1944), paper roll tubes (Gemell and legumes growing in the fi eld. Hartley 2000), or open pots in the laboratory and/or the glasshouse (Bergersen and Turner 1970; Gibson nitrogen fixation in glasshouse 1980). Under these conditions, the N-diff erence and nursery method works well. Th e results of several pot studies using a variety When measuring the amount and rate of N fi xed by of methods of measurement have confi rmed that acacias grown as seedlings in a nursery, or as trees acacias have the capacity for symbiotic N fi xation. in a plantation or forest, the natural abundance Sanginga et al. (1990) showed that fi xation occurred technique appears most appropriate. Th e choice of in 13 provenances of A. albida although at lower tree(s) to be used as reference plant(s) is critical. rates than in leucaena (Leucaena leucocephala). Th ere Of course, they must be non-N-fi xing. Th is is not were diff erences between provenances both in Nfi x usually a problem with temperate species but and %Ndfa, and the two parameters were highly requires some caution with tropical non-legumes, correlated. Using the acetylene reduction assay because some of them may obtain up to 40% of (ARA), Sun et al. (1992a) found that N fi xation, their N requirements from associative N fi xation measured as nitrogenase activity, in young seedlings (Boddey 1987). Th e reference species and the of A. mangium, was linked to the respiration of the test plant should have similar growth rhythms. nodulated roots. Acacia smallii grown at elevated Attention must be paid to sampling procedures. concentrations of CO₂ fi xed more N than plants Ideally, estimates of the absolute amount of Nfi x grown at ambient CO₂ (Polley et al. 1997). Pokhriyal (N fi xation) and of %Ndfa (proportion of whole et al. (1996) noted that nitrogenase activity in plant N, or shoot N, obtained by fi xation of N A. nilotica was highest during the long days of from the atmosphere) should be based on the ratio summer. Th is observation was complemented by Lal of ¹⁵N/¹⁴N of whole plant N or shoot N, not on and Khanna (1993) who showed (in fi eld studies) subsamples of single leaves or other plant parts a decline in N fi xation by A. nilotica during winter (Bergersen et al. 1988). While this is feasible for months. Nitrogenase activity in the nodulated roots sampling seedlings grown in nursery containers, it of A. mangium increased following applications is impossible for trees of the plantation or forest. of phosphorus (P) (Sun et al. 1992b). Likewise, Boddey et al. (2000b) provide a comprehensive Ribet and Drevon (1996) found that low nodule appraisal of all aspects of the natural ¹⁵N abundance nitrogenase activity associated with P defi ciency technique applied to the quantifi cation of biological was linked to reduced nodule growth. On the other N fi xation by woody perennials. While they draw hand, Vadez et al. (1995) concluded that A. mangium

44 nitrogen fixation in acacias seemed not to need high levels of P for growth and N N fi xation. Not all of the investigations quantifi ed fi xation. In A. albida, nitrogenase activity decreased the N fi xed and, even if they had, it would not be after the initiation of water defi cit treatments sensible to extrapolate from pot culture to the fi eld. (Dupuy et al. 1994). Th is fi nding is consistent for Besides, there are numerous fi eld studies, dealt with legumes generally (e.g. Sprent 1971a,b). Aronson below, that have been undertaken for that purpose. et al. (1992) grew 40 legumes, mostly trees, in two Chilean soils. Acacias nodulated and grew better nitrogen fixation in the field than non-acacias and grew more quickly than several species of Prosopis. However, the only evidence that Qualitative evidence N fi xation was responsible for the enhanced growth of the Acacia species was the relationship between Lal and Khanna (1996) reported N fi xation (ARA) rate of growth and extent of nodulation. Ndoye in fi eld-grown A. nilotica, which stopped during et al. (1995) measured N fi xation in A. albida, A. winter. Hansen and Pate (1987a), on the other hand, raddiana, A. senegal and A. seyal. Each species fi xed N found that N fi xation in A. alata and A. pulchella (see Table 4). Th e estimates obtained using diff erent was restricted to the moist months of winter and non-N-fi xing trees as reference (control) plants spring, and essentially ceased during summer and were in reasonably good agreement. A high %Ndfa autumn periods of water stress. Tuohy et al. (1991) did not always lead to high Nfi x. Michelsen and sampled leaves from trees in Zimbabwe. Th ey Sprent (1994) recorded %Ndfa values in A. abyssinica found that leaf N content was consistently higher nursery stock in the range 5–47%. in nodulating tree legumes, including A. nigrescens, than it was in non-nodulating trees of the legume Th ese data make it clear that, grown in pots, all of family Caesalpiniaceae or in non-legumes. Similar the Acacia species examined are capable of symbiotic but less striking data were obtained by Yoneyama

Table 4. Total nitrogen (Nfi x) and proportion of total nitrogen obtained from N fi xation (%Ndfa) calculated for four Acacia species with the ¹⁵N enrichment technique using the non-N-fi xing leguminous trees, Parkia biglobosa and Tamarindus indica, as reference plants.

Acacia species Reference plant P. biglobosa Reference plant T. indica Nfi x (g/plant) %Ndfa Nfi x (g/plant) %Ndfa A. albida 0.4 b* 30.4 b 0.5 b 44.2 b A. raddiana 0.5 b 58.1 a 0.6 b 66.8 a A. senegal 0.4 b 27.2 b 0.5 b 41.6 b A. seyal 1.6 a 59.7 a 1.9 a 66.7 a Source: Derived from Ndoye et al. (1995). * In any one column, values with a common letter are not signifi cantly diff erent from one another (P>0.05).

45 nitrogen fixation in acacias et al. (1993) from leaf samples of trees, including inaccessible to other plants, is a prolifi c fi xer of A. auriculiformis, in Brazil and Th ailand. Following a N (e.g. Heichel et al. 1984; Wivstad et al. 1987; prescribed fi re in a mixed Eucalyptus forest, N fi xed Hardarson et al. 1988; Brockwell et al. 1995b; Gault by the understorey legumes, A. melanoxylon and et al. 1995; Peoples et al. 1995b; Kelner et al. 1997; A. mucronata, increased from near zero after 12 Bowman et al. 2004). months to 26 and 57 mg per plant, respectively, after 27 months (Hamilton et al. 1993). Th is Quantitative evidence observation is contrary to the fi ndings of Hansen and Pate (1987a) who recorded the best N fi xation Investigating alley cropping systems, Sanginga et al. by A. pulchella and A. alata in the fi rst year following (1995) stated that A. mangium grown as hedgerows a controlled burn. might fi x as much as 100–300 kg N per ha per year and A. albida and A. senegal as little as 20 kg N per ha per year. However, these data were not tabulated in their paper. N fi xation of only 5.4 g per tree (Nfi x) was recorded for A. caven grown in a Mediterranean-type climate in Chile over a period of two years (Ovalle et al. 1996). Although fi xed N as a proportion (%Ndfa) represented 85% of total N accumulation), N content was only 1.2% of total biomass production. By comparison, another tree legume, Chamaecytisus proliferus subsp. palmensis (tree lucerne), grown in companion plots produced

Courtesy of CSIRO Forestry and Forest Products Courtesy of CSIRO Forestry and Forest 10 times as much total biomass, although Nfi x Australian acacias are used for many purposes in many and %Ndfa values were similar. May (2001), using countries. Th is 10-year-old plantation of Acacia melanoxylon natural ¹⁵N abundance, measured exceptional N (Tasmanian blackwood) at Gwendique Estate, Zimbabwe, is fi xation by A. dealbata at Tanjil Bren, Victoria, intended for milling. Australia (mean annual rainfall 1900 mm). Over a 5-year period, on land prepared by burning and Robertson (1994) used data obtained from at high stocking rates, the A. dealbata fi xed more modelling levels of soil N in A. senegal/Sorghum than 700 kg N per hectare; about 75% of the N was bicolor rotations to argue that the N-fi xing potential retained in the plant parts, with the remainder in of acacias is less important than their ability to the soil. extract N from deep in the soil profi le. An analogous argument was put for lucerne (Medicago sativa) Further records of N fi xation by acacias in the fi eld (Gault et al. 1991). But it is now recognised that are presented in Table 5. M. sativa, despite having access to deep-soil N

46 nitrogen fixation in acacias

Table 5. Values reported for Acacia nitrogen (N) fi xation in the fi eld Species N fi xation Plant Methodb Period Citation kg/ha %Ndfaa part(s) Acacia albida 2Leaves δ15N natural abundance Schulze et al. (1991) A. tortilis 12 Leaves δ15N natural abundance As above A. hebeclada 15 Leaves δ15N natural abundance As above A. kirkii 17 Leaves δ15N natural abundance As above A. erioloba 21 Leaves δ15N natural abundance As above A. refi ciens 24 Leaves δ15N natural abundance As above A. karroo 25 Leaves δ15N natural abundance As above A. hereroensis 49 Leaves δ15N natural abundance As above A. mellifera 71 Leaves δ15N natural abundance As above A. seyal 63 δ15N natural abundance Ndoye et al. (1995) A. raddiana 62 δ15N natural abundance As above A. pulchella 9-37 Nodules ARAb Hansen and Pate (1987a) A. alata 2-29 Nodules ARA As above A. holosericeac 8-16 Nodules ARA 6 months Langkamp et al. (1979) A. holosericea 6.4 Nodules ARA Annual Langkamp et al. (1982) A. holosericea 4–11 30 Whole plant δ15N enrichment and N 6.5 Cornet et al. (1985)d diff erence months A. dealbata 2–140 Whole plant δ15N natural abundance Per year May (2001) and soil for 5 years A. dealbata 12–32 Nodules ARA Annual Adams and Attiwill (1984) A. mearnsii 200 Whole plant N diff erence Annual Orchard and Darb (1956) A. mearnsii 0.75 Nodules ARA Annual Lawrie (1981) A. melanoxylon 0.01 Nodules ARA Annual As above A. paradoxa 0.04 Nodules ARA Annual As above A. oxycedrus 0.12 Nodules ARA Annual As above A. vernicifl ua 32 Turvey et al. (1983) A. mangium 20–90 Leaves δ15N natural abundance 19 months Galiana et al. (1996) A. longifolia 0.30 Nodules ARA Annual Lawrie (1981) var. sophorae Acacia sp. 52–66 Whole plant δ15N natural abundance Peoples, Almendras and Darte

47 nitrogen fixation in acacias

Table 5. (cont’d) Values reported for Acacia nitrogen (N) fi xation in the fi eld Species N fi xation Plant Methodb Period Citation kg/ha %Ndfaa part(s) Acacia sp. 51-81 Whole plant δ15N natural abundance Dart and Almendrase Acacia sp. 84-88 Whole plant δ15N natural abundance Dart and Almendrase Acacia sp. 34-67 Whole plant δ15N natural abundance Dart and Almendrase Acacia sp. 69 Whole plant δ15N natural abundance Palmere Acacia sp. 56 Whole plant δ15N natural abundance Palmer and Tatange Acacia sp. 59 Whole plant δ15N natural abundance Palmer and Tatange a %Ndfa is the proportion (%) of total N in the plant (or plant part) derived from the atmosphere by symbiotic N fi xation. b ARA = the acetylene reduction assay. c Originally identifi ed as Acacia pellita O. Schwarz — Langkamp et al. (1979), then Corrigendum (1980). d Trees grown in containers of 1 m3 volume, not in the fi eld. e Unpublished data.

Measurements for other tree legumes are given in methodology. Perhaps the fi gure quoted by Orchard Table 6. Th e most consistent feature of the diverse and Darb (1956) — 200 kg N fi xed per ha per measurements of N fi xation by acacias is their year — was on the high side of reality because the inconsistency. Values ranged from 0 to 200–300 kg N diff erence method that they used has a tendency N per ha per year, but were generally at the lower towards overestimation. It is not a simple matter, end of that scale. Patterns of N fi xation over time either, to reconcile the promising values for acacia were sometimes contradictory. A remarkable N fi xation obtained in glasshouse and nursery discrepancy between the growth of acacias in with the generally low values from the fi eld. In the fi eld and in the glasshouse was reported by the next section, we consider factors that might Hansen and Pate (1987a). Th ey compared symbiotic aff ect N fi xation performance of fi eld-growing seedlings of A. alata and A. pulchella regenerating in a acacias. Environmental factors and symbiotic forest ecosystem with seedlings of the same species, factors are considered separately. Symbiotic factors inoculated with forest soil containing naturally are deemed to include relationships between occurring strains of acacia rhizobia, growing in acacias and mycorrhizae as well as associations N-free medium in the glasshouse. By 19 months, the between acacias and rhizobia. Later, we will also glasshouse plants had gained 130–230 times more consider what implications those factors might dry weight and had accumulated 110–160 times have in relation to optimisation and exploitation more total N than seedlings in the forest. of N fi xation in acacias in order to enhance the N nutrition and vigour of the tree, to conserve soil N We do not believe that the variability in results from and to contribute to the sustainability of forest and the fi eld can be attributed solely to measurement plantation ecosystems.

48 nitrogen fixation in acacias

Table 6. Estimation of nitrogen (N) fi xation in leguminous trees other than species of Acacia

Genus N fi xation Period Citation kg/ha %Ndfaa Aotus ericoides 1 Annual Lawrie (1981) Albizzia 94 60 Annual Liya et al. (1990) Albizzia 55 Peoples, Almendras and Dartb Calliandra 11 14 90 days Peoples and Palmerb Gliricidia 108 72 Annual Liya et al. (1990) Gliricidia 13 Annual Roskoski et al. (1982) Gliricidia 99 75 Annual Peoples and Palmerb Gliricidia 60 Annual Peoples and Ladhab Inga 35 Annual Roskoski (1981) Leucaena 110 Annual Hogberg and Kvarnstrom (1982) Leucaena 296–313 58–78 3 months Zoharah et al. (1986) Leucaena 288–344 34–39 6 months Sanginga et al. (1989b) Leucaena 59–100 Yoneyama et al. (1990) Source: derived from Peoples and Craswell (1992), Khanna (1998) and unpublished data. a %Ndfa is the proportion (%) of total N in the plant (or plant part) derived from the atmosphere by symbiotic N fi xation. b Unpublished data. environmental factors affecting moisture impose limitations on the host (cf. Th ies et nitrogen fixation in the field al. 1991a,b; Peoples et al. 1995a). Giller and Wilson (1991), dealing with N fi xation by leguminous trees General considerations and shrubs as well as by agricultural legumes in A principle of limiting factors states that ‘the tropical settings, presented a comprehensive review level of crop production can be no higher than of environmental constraints to N fi xation. that allowed by the maximum limiting factor’. If a similar principle applied to symbiotic N fi xation by In the previous section, we showed that acacias acacias, it would follow that the level of N fi xation fared poorly in N fi xation by comparison with other would be strongly linked to the physiological state leguminous trees (Tables 5 and 6). However, a direct of the host tree. However eff ective an association comparison of the two groups may not be valid. between an acacia and a rhizobial strain might be, Nitrogen-fi xation studies of tree legume genera such it cannot realistically be expected to express its full as Calliandra, Gliricidia and Leucaena were mostly potential for N fi xation if limiting factors such as done on trees planted in hedgerows, often fertilised nutrient defi ciency or excess, salinity, unfavourable and inoculated with eff ective root-nodule bacteria, soil pH, soil microbiology, and/or insuffi cient soil and intended for periodic harvesting and grazing

49 nitrogen fixation in acacias of foliage. On the other hand, much of the work on abundance to measure the proportion of leaf N due acacias was done in natural habitats — savannas, to N fi xation in arid environments (30–400 mm sand dunes, mixed-species forests. annual rainfall) in Namibia. Th ey recorded values ranging from 2% Ndfa for A. albida to 71% Ndfa An apt analogy is subterranean clover (Trifolium for A. mellifera (see Table 4). Th ey were unwilling, subterraneum), which is a legume component of however, to attribute their results solely to N some 38 million hectares of pasture in southern fi xation, speculating that deep-rooted species may Australia and a prolifi c fi xer of N. It is an have accessed soil that was highly enriched with insignifi cant species in its native habitat on the ¹⁵N (cf. Virginia et al. 1989). It was not mentioned mainly acid, phosphorus-defi cient soils of the whether the roots of the trees also had access to Mediterranean basin. Indeed, for 50–80 years after groundwater. Robertson (1994), modelling data its accidental, but continuing, introduction into obtained from an arid-zone system, concluded that Australia, it remained a plant of little importance. It A. senegal had limited potential for symbiotic N was not until the early 1900s, when superphosphate fi xation. Newton et al. (1996) regarded out-planted began to be used, and when other nutrient element A. tortilis subsp. spirocarpa as a tree with a relatively defi ciencies were corrected later, that subterranean high water-use effi ciency that, in some situations, clover responded and began its rise to prominence might be further improved by rhizobial inoculation. as a pasture plant (Morley and Katznelson 1965). Barnet et al. (1985), working with A. longifolia var. Even then it fl ourished only in soils where eff ective sophorae, A. suaveolens and A. terminalis growing strains of rhizobia occurred. It is apparent that on sand dunes, found that N fi xation maximised in the success of subterranean clover in Australia is a late autumn when moisture was readily available consequence of good agronomic management. We and declined in late spring and early summer as submit that effi cient silvicultural management will moisture became limiting and nodule senescence be the key to realising the full potential of acacias increased. Seasonal changes in nodulation that lead for production of wood and symbiotically fi xed N. to substantial variations in rates of N fi xation are However, before that potential can be exploited, characteristic of Australian native legumes and have the factors that currently limit productivity and N been recorded over a long period for a number of fi xation must be defi ned. species across a wide geographic range (e.g. Beadle 1964; Langkamp et al. 1981, 1982; Lawrie 1981; Monk et al. 1981; Hingston et al. 1982; Hansen Soil moisture and Pate 1987a,b; Lal and Khanna 1996). Deans Habish (1970) regarded 15% soil moisture as et al. (1993) worked with a soil from Sudan that optimal for growth and nodulation of Acacia species contained large populations of acacia rhizobia but generally. Information about the relationship in which A. mellifera trees did not form nodules. between N fi xation in acacias and soil moisture is Th ey attributed the lack of nodulation to low soil contradictory. Schulze et al. (1991) used natural ¹⁵N moisture.

50 nitrogen fixation in acacias

Th ere seems no reason why the eff ect of moisture Many acacias grow in hot climates where, at certain stress on N fi xation by acacias should be any times of the year, surface soil temperatures are diff erent from what was demonstrated in the high enough to prevent N fi xation altogether classical work of Sprent (1971a,b) working with the (cf. Gibson 1971). However, it is distinctly possible nodules of soybean. Whenever and wherever water that deep-rooted legumes such as acacias may escape is defi cient, legume N fi xation will be impaired. extremes of environment by forming the bulk of Should the defi ciency be severe, N fi xation will their nodules (and fi xing most of their N) at depth, cease. It is also probable that acacias, like other where conditions are more benign. An analogous legumes, will shed nodules (Sprent 1971a; Sheaff er circumstance has been reported from a hot climate in et al. 1988) as well as other below-ground parts Australia. In a 3-year-old stand of lucerne (Medicago during periods of great moisture stress. sativa), more than 99% of the total soil population of lucerne rhizobia (Sinorhizobium meliloti) congregated at 30–60 cm depth in the soil profi le; presumably, Soil temperature the majority of nodulation and N fi xation took place Th ere appears to be little recent published in the same vicinity (Evans et al. 2005). information on the eff ects of temperature on acacia nodulation and N fi xation. However, there is no Light reason to suppose that infl uences of low and high temperatures on acacia symbioses would diff er very Th ere is substantial evidence that low levels of light much from temperature eff ects on the symbioses restrict the growth of legumes [references cited by of forage legumes (e.g. Gibson 1963, 1969, 1971; Sprent (1999)] due, at least in part, to deleterious Harding and Sheehy 1980). eff ects on N fi xation. Th is may be of signifi cance for acacias growing as forest understorey. In Australia, Habish (1970) found that Acacia species produced acacias are often the dominant recolonising eff ective nodules at temperatures up to 35°C, species following bushfi re and may fi x abundant N which he considered ‘the highest temperature for during this phase (e.g. Adams and Attiwill 1984; nodulation so far recorded’. Rasanen and Lindstrom Hansen and Pate 1987a,b). In the following phase (1999) studied the eff ects of high temperature on of succession, as species of Eucalyptus become rhizobial infection of acacia root hairs, which is dominant, the acacia understorey becomes sparse an early step in the processes leading to nodule and obviously less eff ective in fi xing N. Competition formation. Th ey found that infection and nodulation for light may contribute to the condition. Roggy and were normal at (high) temperatures below 38°C but Prevost (1999) recorded nodulation of both shade- that nodulation was reduced at 38° and 40°C and tolerant and shade-intolerant legume trees growing ceased completely at 42°C. in tropical forests but did not measure relative levels of N fi xation.

51 nitrogen fixation in acacias

Nutrients Th e literature is somewhat confusing about the Th ere are several comprehensive reviews of response of acacias to fertiliser. Although acacias the nutrient needs of legumes generally, both are generally adapted to soils of low fertility, many dependent on and independent of biological N (tropical) species respond to the application of fi xation (e.g. Vincent 1965; Munns 1977; Smith fertilisers (e.g. Cole et al. 1996). In particular, 1982; O’Hara et al. 1988; Giller and Wilson 1991). response to added phosphorus (P) is common, Information on the requirements of acacias, on and benefi ts have also been obtained by applying the other hand, is sparse. For symbiotic legumes, micro-nutrients and mineral N (Mead and Miller the mineral requirements of the rhizobia, the 1991; Goi et al. 1992; Turvey 1995; Cole et al. infection process, nodule development and nodule 1996). Generally, early growth of nursery seedlings function are usually less than for the plant itself. is increased by adding mineral N as a starter, but But there are important exceptions — references low levels of nitrate depress nodule formation cited by Brockwell et al. (1995a). For instance, by A. auriculiformis (Goi et al. 1992). Plantation for nitrogenase (N-fi xing) activity, molybdenum trees respond better to fertiliser added at planting and cobalt are needed far in excess of other plant than to applications after canopy closure (Otsamo requirements (Evans and Russell 1971). Also, 1996). In A. mangium, there is a link between low P evidence is accumulating (McLaughlin et al. 1990) and poor N fi xation (Mead and Speechly 1991) as of a specifi c eff ect of P on the growth and survival there is with herbaceous crop and pasture legumes of rhizobia and their capacity for nodulation and (cf. Vincent 1965). On the other hand, Ryan et al. eff ective N fi xation (Singleton et al. 1985). (1991) reported relatively small, albeit signifi cant, responses to the application of a complete Critical foliar levels for the nutrient elements mineral fertiliser including N by newly outplanted that may limit the growth of acacias are yet to be A. neriifolia, A. cincinnata, A. leptocarpa, A. mangium, determined (Simpson et al. 1998). However, while it A. crassicarpa and A. plectocarpa. A curious side- is quite obvious that there are substantial diff erences eff ect was that survival of seedlings was depressed in nutritional requirements between Acacia species, signifi cantly by addition of fertiliser. Th is appeared it is also clear that acacias are less demanding of to be due primarily to P, but trace elements were nutrients than many agricultural legumes. Th is implicated also. character is exploitable. For instance, vast areas of degraded, infertile, Imperata grasslands in Indonesia Lesueur and Diem (1997) showed that the are scheduled for reforestation with A. mangium A. mangium/Bradyrhizobium association had a high (Turnbull et al. 1998b). Th is is not to say that acacias and ongoing requirement for exogenous iron to are immune to mineral defi ciency. Dell (1997) found promote nodulation and maintain N fi xation. Th eir boron and iron defi ciencies, and nickel toxicity, in A. results were interpreted to mean that A. mangium mangium growing on very infertile acid soils in parts lacked mechanisms to store phytoferritins in its of China, the Philippines and Indonesia. tissues.

52 nitrogen fixation in acacias

In considering the sometimes confl icting results of It is very likely that NO₃– also inhibits the investigations relating to nutrition, we concur with nodulation and N fi xation of Acacia species. Turk the views of Dommergues (1982) who concluded et al. (1993) considered that plant-available N in that the productivity of N-fi xing trees and their soil used in pot experiments reduced the response ability to fi x N were dependent, amongst other to inoculation by A. auriculiformis, A. mangium and things, on nutrient status. Likewise, Jordan (1985) A. mearnsii. Th e degree of inhibition seems bound to considered that nutrient defi ciency was the major vary with circumstance. Toky et al. (1994) observed factor limiting forest productivity in the humid that application of urea reduced nitrogenase activity tropics. in nodulated A. nilotica without any corresponding eff ect on the extent of nodulation. Likewise, Th akur Experimental acacia plantations in Australia et al. (1996) found that short-term (5–15 days) are typically (Mitchell 1998; Searle et al. 1998) application of nitrate to seedlings of A. catechu but not always (Bird et al. 1998) supplied with had no eff ect on nodulation. In plantation forests, superphosphate and sometimes nitrogenous establishment of acacia seedlings usually follows fertiliser at planting and/or at 12 and 24 months land preparation procedures that result in release thereafter. By contrast, acacias established by of substantial amounts of NO₃– from organic direct seeding in land reclamation undertakings sources. Th e presence of such N is ephemeral due to (see below) in southern Australia are rarely, if ever, leaching and other losses, and the element is soon supplied with mineral fertiliser; they are, however, unavailable to the developing seedlings. Th erefore, inoculated with strains of rhizobia to provide a fertiliser N is often applied at or soon after planting source of biological (fi xed) N. (e.g. Searle et al. 1998; Mitchell 1998). We believe that outplanting Acacia seedlings that are well nodulated and actively fi xing N would remove any Nitrate requirement for N fertiliser at planting. Inhibition of nodulation and N fi xation by nitrate (NO₃–) is well known (Vincent 1965; Peoples In natural forest ecosystems and long-established and Herridge 1990). Th e processes are not fully plantation systems with a substantial component understood, although several hypotheses have of N-fi xing trees, leaf litter on the forest fl oor is been proposed (e.g. Tanner and Anderson 1964; relatively rich in organic N. As this is broken down, Munns 1968; Dazzo et al. 1981). Th ere are serious the soil profi le immediately beneath the litter layer implications for leguminous fi eld crops and their is likely to become enriched in plant-available N capacity to form eff ective symbioses (e.g. Herridge as NO₃–. Due to higher rates of N cycling, N is et al. 1984; Bergersen et al. 1985; Th ies et al. more readily available in natural (tropical forest) 1991b). Indeed, Brockwell et al. (1995a) concluded ecosystems than in anthropogenic systems (e.g. that it was unwise to plant a legume into soils Neill et al. 1999; Rhoades and Coleman 1999; containing signifi cant amounts of available NO₃–. Veldkamp et al. 1999; Paparcikova et al. 2000).

53 nitrogen fixation in acacias

Th is has implications for the level of N fi xation A. auriculiformis, but not their rhizobia, for tolerance achieved by those N-fi xing trees whose general to zinc as a prerequisite for using the species for nutrition is heavily reliant on surface-feeder roots. revegetation of zinc-contaminated land. It also has signifi cant implications for the N status of similar non-N-fi xing trees or other species that Landfi ll gas might be used as reference plants as part of the natural ¹⁵N abundance technique for measuring In Hong Kong, A. confusa and leucaena (Leucaena symbiotic N fi xation (Boddey et al. 2000b). leucocephala) are widely used for revegetating landfi ll areas. Gaseous emissions from the landfi ll include oxygen, methane, carbon dioxide and acetylene. Heavy metals Prolonged exposure to landfi ll gases suppressed the Th ere is little doubt that the introduction of heavy growth and N fi xation of these species (Chan et al. metals by application of sewage sludge or spoil from 1998). mining operations reduces the size and diversity of populations of soil microfl ora, including rhizobia Soil salinity (e.g. McGrath et al. 1988; Chaudri et al. 1992, 1993; Angle et al. 1993; Dahlin et al. 1997; Giller et al. A number of Acacia spp. tested in the fi eld have been 1999). It has also been suggested that heavy metals shown to have varying degrees of tolerance of saline may impair the eff ectiveness of N-fi xing populations soils (Aswathappa et al. 1986; Marcar et al. 1991a, (Smith 1997). Th ose of the reports that deal with 1998). Amongst the most tolerant are A. ampliceps, rhizobia focus mostly on R. leguminosarum bv. A. nilotica, A. redolens, A. saligna and A. stenophylla. trifolii. Th ere are diff erences between provenances of the same species (Craig et al. 1991; Marcar et al. 1991b, It has been argued that the extent of heavy metal 1998). impairment of microfl oral populations is a function of soil pH and organic matter (Brendecke et al. Marcar et al. (1991a) demonstrated in sand-culture 1993; Pepper et al. 1994), but there has been much solution augmented with three levels of sodium debate on this (Pepper et al. 1994; Witter et al. chloride (NaCl) that A. ampliceps, A. auriculiformis 1994a,b; McGrath and Chaudri 1999). and A. mangium, in that order, were progressively less tolerant of salinity. Sodium chloride We are unaware of any published information on concentrations of 400 mM markedly aff ected shoot eff ects of heavy metals on the rhizobia of acacias. dry matter, nodule number and nodule development However, some workers apparently take the view in all three species. Tree species with artifi cially that Acacia species are more sensitive to heavy high levels of foliar N, induced by applications metals than are their rhizobia. For instance, Zhang of ammonium nitrate, generally had lower shoot et al. (1998) recommended screening lines of concentrations of sodium and calcium than

54 nitrogen fixation in acacias inoculated plants wholly dependent on symbiotic N. salt-sensitive, to inoculate A. ampliceps grown at Otherwise, survival, damage symptoms and relative increasing concentration of NaCl. Indices of N growth reduction as salt concentrations increased fi xation — nodule number, acetylene reduction, N were similar for both normal and high N treatments. content per plant, plant growth — declined as salt concentration increased, but the decline was less Zhang et al. (1991) considered that rhizobia from where the A. ampliceps plants had been inoculated the nodules of trees, including acacias, growing in with the salt-tolerant strain than where they had Sudan had a high tolerance of salt. A strain isolated been inoculated with the salt-sensitive strain. Craig from A. farnesiana tolerated salt concentrations up et al. (1991) worked with two rhizobial strains to 5% (Surange et al. 1997). Two of 35 rhizobial isolated from A. redolens growing in saline areas and isolates from A. nilotica grown in culture medium one each from A. cyclops and A. saligna growing in tolerated up to 850 mM NaCl (Lal and Khanna non-saline soils. All four were equally tolerant of 1994). However, when those two strains were buff ered culture medium to which 300 mM NaCl inoculated on to A. nilotica grown under saline had been added. Th ree of the strains, including conditions, they lost at least 75% of the N-fi xing both from the saline areas, when combined with (nitrogenase) activity that they had exhibited when a highly salt-tolerant provenance of A. redolens grown in combination under non-saline conditions. formed associations that did not diff er in symbiotic In glasshouse experiments, Zou et al. (1995) used characteristics irrespective of NaCl concentration two strains of rhizobia, one salt-tolerant and one up to 160 mM. On a less salt-tolerant provenance rall Peter Th Peter Severely salted landscapes cannot be rehabilitated but their expansion can be contained by revegetation around their perimeters. Salt-tolerant species of Acacia are apt for this purpose.

55 nitrogen fixation in acacias of A. redolens, and on A. cyclops, the infectivity Soil reaction — acidity and N-fi xing eff ectiveness of the strains fell as the Th ere is scant literature on acacia tolerance of soil external salt concentration increased. Cantrell acidity. Th is may be because there is a complex of and Linderman (2001) reported that eff ects of soil factors involved in soil acidity, so that screening salinity on two non-legumes were ameliorated by plants for tolerance of acidity is more complicated pre-inoculation with arbuscular mycorrhizal fungi. than screening them for salt tolerance. Habish (1970) considered that soil reaction aff ected acacia It appears from these investigations that the acacia nodulation more than it aff ected plant growth. At root-nodule organism is less sensitive to external pHwater 5.0–5.5, nodulation was absent. Lesueur et NaCl concentration than the acacia plant, and that al. (1993) recognised that A. albida and A. mangium the acacia symbiosis is at least as sensitive as the plants, in contrast to acacia rhizobia, might be acacia plant. We concur with Bala et al. (1990) that aff ected by acidity (soil pH 4.5), depending on it is sensible to use eff ective, salt-tolerant strains plant provenance. Although aluminium toxicity in of rhizobia to inoculate acacia species intended agricultural species is a common phenomenon in for planting on saltlands. Under these conditions, acid soils (e.g. Delhaize and Ryan 1995; Ma et al. one can be confi dent that, provided that the plant 2001), Lesueur et al. (1993) could detect no eff ect of survives, so too will the bacteria. high concentrations of aluminium (100 mM AlCl₃) on the growth of A. albida or A. mangium. Th ey nevertheless considered that the ability of acacias to tolerate soil acidity should be taken into account when screening Acacia/Bradyrhizobium combinations for use in aff orestation trials. Searching for acid- tolerant woody legumes suitable for alley cropping, Kadiata et al. (1996) eliminated A. auriculiformis as a candidate. Acacia mangium had a high rate of N fi xation (measured as acetylene reduction) in acid soil in Costa Rica (Tilki and Fisher 1998). Snowball and Robson (1985) observed that A. signata, a species adapted to acidic soil, responded to application of lime. Th is was thought to be due

Courtesy of CSIRO Forestry and Forest Products Courtesy of CSIRO Forestry and Forest to alleviation of manganese toxicity. Ashwath et al. Many acacias are substantially tolerant of salinity (1995) used glasshouse experiments with two soil (Aswathappa et al. 1986). One such species is Acacia types to rank 36 symbiotic Acacia species according stenophylla (Eumong or river cooba). Th is fi ne specimen to acid tolerance. Ranking diff ered somewhat of A. stenophylla is growing on a saline sandy soil near with soil type. Acacia julifera, A. aneura, A. diffi cilis Oodnadatta, South Australia. and A. tumida were ranked as highly tolerant or

56 nitrogen fixation in acacias tolerant on both types. Th e highly salt-tolerant Collembola (non-insectan arthropods, springtails) species, A. stenophylla, was very sensitive to acidity. in forest soils, Pinto et al. (1997) concluded Other results from the experiments indicated that that the presence of acacias contributed to acacias are likely to benefi t from inoculation with increases in the population densities of soil fauna. eff ective rhizobia, particularly when grown on acidic Synergistic phenomena such as these are probably soils. Kang et al. (1998) observed that most acacia commonplace and are mediated also by non- rhizobia are sensitive to acidity but that strongly leguminous trees (e.g. Hansen 1999). acid-tolerant strains do exist. Parasites and pests Soil reaction — alkalinity Like most other plants, acacias are susceptible to Tolerance of alkaline conditions by certain acacia predation by nematodes, e.g. Meloidogyne spp. rhizobia is also common and occasionally quite (Duponnois et al. 1997a,b). Th e attacks are generally extraordinary. For instance, a strain isolated from directed at the roots, and it seems reasonable to A. farnesiana was well adapted to grow at pH 12.0 suppose that the symbiotic system would sustain (Surange et al. 1997). Habish (1970), on the other collateral damage. Robinson (1961) and Taha and hand, speculated that the alkaline reaction of soils Raski (1969) report nematode invasion of the in northern Sudan might be a factor in reducing nodules of agricultural legumes. Duponnois et (nodulation and) plant growth of acacias in that al. (1997a) observed that M. javanica infestation region. reduced N fi xation by A. mangium and A. holosericea. Acacia tumida and A. hilliana, on the other hand, fi xed more N in the presence of the nematode. Cluster roots

Many plants, including acacias, have cluster Like other plants, acacias are subject to to (proteoid) roots, which are one of several means by infestation by mistletoes (e.g. Olax spp.), which plants cope with inhospitable environments hemiparasites (e.g. Santalum spp.) and various (Sprent 1999). Cluster roots may be important strangler vines. Ku et al. (1997) and Tennakoon in N acquisition, supplementing N fi xation et al. (1997a,b) investigated some consequences (Sprent 1995), as well as facilitating uptake of of the parasitic association between nodulated other nutrients — e.g. iron (Waters and Blevins A. littorea and Olax phyllanthi. Parasitism had no 2000) — and soil moisture (Sprent 1995). eff ect on the total increment of N fi xed. However, partitioning within the acacia plant of the products of N fi xation was disturbed, which led to decreased Soil fauna shoot biomass and total plant N but increased root Th e density of soil fauna is sometimes regarded growth. About 9% of the haustoria of the mistletoe as an index of ‘soil health’. Based on studies of was attached directly to the root nodules of the

57 nitrogen fixation in acacias acacia which almost certainly provided the parasite indigenous fl ora. Acacia smallii (now A. farnesiana with immediate access to the products of the N var. farnesiana) was recorded as a serious invader fi xation. Th e mistletoe also acquired some fi xed of grassland (Polley et al. 1997); it is not known N from the xylem sap of the acacia (Ku and Pate if its N-fi xing ability was a factor. In Australia, 1997; Tennakoon and Pate 1997). Similarly, the root native acacias such as A. baileyana and A. longifolia hemiparasites, Santalum spp., acquired N from the sometimes become weeds. xylem sap of A. ampliceps and A. trachycarpa hosts (Tennakoon et al. 1997a; Radomiljac et al. 1998).

Two insect species, Sitona sp. and Rivellia sp., are known to attack legume nodules (Diatloff 1965; Gibson 1977), and might thus aff ect N fi xation. Diatloff (1965) reported that Rivellia larvae damaged 50–70% of nodules on perennial glycine — Glycine (now Neonotonia) wightii, but there is no record of these insects infesting acacia nodules.

Like other plants, acacias are subject to attack

by insects — see e.g. Searle (2000) — and fungal Brockwell John pathogens — see e.g. Old (1998). It is not known Acacia baileyana (Cootamundra wattle) makes an attractive what eff ects such attacks have on symbiotic N ornamental for parks and gardens. Unfortunately, it may fi xation. However, it is a rule of thumb with legumes become an invasive weed of bushland outside its natural that any factor that reduces plant growth also environment. lessens N fi xation (cf. Peoples et al. 1998; Peoples and Baldock 2001). mycorrhizal factors affecting nitrogen fixation in the field Acacias themselves may attain pest status — see e.g. Holmes and Cowling (1997). Acacia dealbata, Many plant species benefi t from associations an Australian native species, is a serious weed in with mycorrhiza, mainly because of the ability of Chile, India and South Africa due to its prolifi c the fungi to act as conduits for plant nutrients, seed production and suckering habit (Doran and scavenged from infertile soil, that would be Turnbull 1997). Stock et al. (1995) reported that the otherwise inaccessible to the plant. Th e associations N fi xation of alien species A. cyclops and A. saligna tend to be of greatest benefi t to the plant under was a signifi cant factor in their invasion of two conditions of low fertility. Indeed, even moderate South African ecosystems and their establishment, amounts of fertiliser — e.g. phosphate (Kahiluoto persistence and successful competition with et al. 2000) — depress growth of mycorrhiza and

58 nitrogen fixation in acacias reduce the infectivity and effi ciency of the fungi. In (1992) and Mandal et al. (1995) demonstrated agroforestry, mycorrhizal associations contribute growth responses in A. nilotica to co-inoculation much to the growth of Acacia species in unfertilised with rhizobia and mycorrhizal fungi, but did not fi elds (Dart et al. 1991). Reddell and Warren (1987) examine the eff ect of each organism individually. listed nearly 50 species of Acacia with mycorrhizal Lal and Khanna (1996) noted that the growth of associations. Some aspects of the management of A. nilotica after joint inoculation with one rhizobial mycorrhizas in forestry have been dealt with by strain and Glomus fasciculatum was better than after Grove and Malajczuk (1994) and Jasper (1994). inoculation with either organism individually. Th is Reddell and Warren (1987) drew attention to the synergism appeared to be a specifi c eff ect involving potential for using inoculants of mycorrhizal fungi that particular strain of rhizobium rather than a to improve the survival, establishment and growth general phenomenon. Ba et al. (1994) observed that of tropical acacia plantations. Th ey speculated that the ectomycorrhiza, Pisolithus tinctorius, interfered nursery inoculation of seedling stock destined for with rhizobial infection thread development outplanting into the fi eld might be an effi cient and nodule meristem initiation in A. holosericea. means of fulfi lling this potential. Th ese results were obtained in axenic culture in the laboratory. It seems unlikely that they would Acacias form associations with both endomycorrhiza extrapolate to the complex microfl oral conditions of and ectomycorrhiza (Reddell and Warren 1987). the fi eld. Endomycorrhiza — commonly known as vesicular- arbuscular mycorrhiza (VAM) — invade the Using the natural abundance technique, Michelsen roots. Ectomycorrhiza colonise the root surfaces. and Sprent (1994) found that some vesicular- Acacias respond to inoculation with either type arbuscular mycorrhiza improved N fi xation by (e.g. Dela Cruz and Yantasath 1993; Osunubi et four Acacia species growing in a nursery, although al. 1996; Munro et al. 1999). Some Australian there was no corresponding increase in shoot N acacias associate with both types of mycorrhiza concentration. Franco et al. (2001) considered that and form root nodules (Sprent 1994b) as well. joint inoculation of tree legumes with rhizobia While endomycorrhiza occur frequently in soils and mycorrhiza held promise as an aid to land growing acacias, ectomycorrhiza are less common reclamation in the humid Amazon. Chung et al. and may be absent from some soils (Khasa et al. (1995), on the other hand, found no benefi t from 1994). Combined mycorrhiza and root-nodule co-inoculation. Th is work was done with hybrid bacteria (cf. Mosse et al. 1976) may synergistically plantlets from tissue culture. Acacia confusa stimulate N fi xation in legumes growing in soil and A. mangium in pot experiments responded that is defi cient in plant-available P; for example, to dual inoculation with vesicular-arbuscular Dela Cruz and Yantasath (1993) noted enhanced mycorrhizal fungi and phosphorus-solubilising growth of A. mangium following inoculation with bacteria (Young 1990); the Acacia species may not both mycorrhiza and rhizobia. Beniwal et al. have been nodulated. Ba et al. (1996) considered

59 nitrogen fixation in acacias that the use of ectomycorrhiza can contribute rhizobial factors influencing to an increase in the N-fi xing potential of nitrogen fixation in the field A. holosericea and A. mangium and were optimistic General considerations that endomycorrhiza might have the same value. Th ese views mirror our own conclusions about the Th ere are always three aspects of the legume association between mycorrhiza and N fi xation by symbiosis to consider when appraising factors acacias generally. In some instances, the tripartite that aff ect the effi ciency of N fi xation: the plant, relationship including rhizobia can be synergistic the bacteria and the interaction between them. but the conditions required for that to occur are not Variation can and does occur in each of these well defi ned. aspects. It is particularly signifi cant in the host plant and in the rhizobia, and has major implications for A more subtle infl uence on the N economy of the establishment of a symbiosis that is eff ective in acacias appears to lie in the ability of mycorrhiza to fi xing N. access multiple forms of N from the soil. Whereas plants, including trees (Devisser and Keltjens Genetic variability of host acacias 1993; Turnbull et al. 1995) such as acacias, are largely restricted to the use of nitrate (NO₃–) and During an examination of more than 40 lines of ammonium (NH₄+) from the soil and, for legumes, A. nilotica, Beniwal et al. (1995) and Toky et al. N from the atmosphere, mycorrhiza is more (1995) recorded marked genetic variation among acquisitive. Th ere is now abundant evidence, cited provenances in ability to fi x N. A similar study by Boddey et al. (2000b), that mycorrhizal fungi by Burdon et al. (1998) of 67 populations of 22 and the plants that they infect are able to absorb Acacia species and associated strains of rhizobia from the soil, in addition to NO₃– and NH₄+, amino from south-eastern Australia found little evidence acids and N from proteins and chitin. Dommergues of rhizobia strain/Acacia provenance eff ects on (1982) was suffi ciently impressed with the potential N fi xation. Th ey concluded that elite rhizobial benefi ts of mycorrhizal infection to N-fi xing trees cultures from one provenance would perform to suggest that, as well as inoculation with rhizobia, well on other provenances. However, signifi cant ectomycorrhizal and endomycorrhizal inoculants host-based variation in the capacity to form should be considered. It is of interest to note that eff ective symbiotic associations was detected in Cantrell and Linderman (2001) observed that pre- half-sib families of A. dealbata, A. mearnsii and inoculation of lettuce (Lactuca sativa) and onion A. melanoxylon. Th is led to the suggestion that, (Allium cepa) with endomycorrhizal fungi reduced in acacia breeding programs, it would be prudent harmful eff ects of soil salinity. It is not known to continually monitor the N-fi xing capacity of whether mycorrhizas confer similar benefi ts on breeding material. Similarly, Sun et al. (1992c) used acacias growing in saline environments. rhizobial inoculation and application of combined N to study the symbioses of multiple provenances

60 nitrogen fixation in acacias of A. auriculiformis, A. mangium and A. melanoxylon. other group, the so-called cowpea miscellany, for Signifi cant variation in nodulation and responses about 19,000 species across all legume families, to applied N led them to conclude that there was Mimosaceae, Caesalpiniaceae and Fabaceae; (ii) scope to increase growth by plant selection. Sprent single tribes (Trifolieae, Phaseoleae) are represented (1995) also considered that there was substantial in more than one cross-nodulation group; (iii) potential to enhance acacia symbiosis by plant genera in the same cross-nodulation group (Lupinus, selection. Zorin et al. (1976) demonstrated with Ornithopus) belong to diff erent tribes; (iv) the Caucasian clover (Trifolium ambiguum), growing cowpea group is clearly ‘miscellaneous’, extremely axenically in tubes of vermiculite moistened with diverse and merely a ‘catch-all’. By implication, McKnight’s nutrient solution (McKnight 1949), that Acacia was allocated to the cowpea group. Th is was a meaningful preliminary selection for increased N accurate in that rhizobia isolated from Acacia spp. fi xation could be achieved within 28 days. Like T. formed nodules, and often fi xed N, with Vigna ambiguum, species of Acacia are poorly domesticated, unguiculata (cowpea). vary in symbiotic response from plant to plant (Burdon et al. 1998, 1999) and can be grown under bacteriological control (Kang et al. 1998). Th erefore, the procedure might feasibly be used among acacias and their rhizobia for selecting for enhanced symbiosis.

Cross nodulation

Th e concept of cross nodulation, a manifestation of host/rhizobial specifi city, also known as cross inoculation, was developed in early studies of the symbiosis between legumes and root-nodule bacteria (Fred et al. 1932). Th e concept dictates

that related groups of legumes are nodulated by Alison Jeavons particular rhizobia, and that those rhizobia will Many strains of rhizobia that nodulate Acacia spp. are often nodulate across all legumes in the group (cross- highly promiscuous in that they also form nodules, and may nodulation group) but will not nodulate plants in fi x nitrogen, with many other species including those from other cross-nodulation groups. Th e classical version diff erent families. Th is phenomenon is an example of ‘cross- of the system of cross-nodulation groupings is nodulation’. Th e Australian native shrub, Daviesia ulicifolia portrayed in Table 7. Th ere are clear anomalies in (gorse bitter pea) — family Fabaceae, cross-nodulates with the system: (i) the groupings are selective, with six Acacia spp. — family Mimosaceae. groups accounting for about 1000 species and the

61 nitrogen fixation in acacias

Despite all its anomalies, the concept of cross leguminosarum bv. trifolii strain TA1 was, and still nodulation had practical value. It provided a is, the sole rhizobial component of commercial convenient guide for manufacturers of commercial inoculant for seven species of Trifolium: legume inoculants used in agriculture because it T. alexandrinum (berseem clover), T. dubium allowed them to use a single strain of rhizobia, (suckling clover), T. fragiferum (strawberry clover), and therefore a single inoculant, for a number of T. glomeratum (cluster clover), T. hybridum (alsike diff erent legumes of the same cross-nodulation clover), T. pratense (red clover) and T. repens (white group. In Australia, for instance, Rhizobium clover) (Brockwell et al. 1998).

Table 7. Portrayal of an early version of the system of legume cross inoculation (cross nodulation) grouping

Group Genus No. of speciesa Tribe Family Root-nodule bacteriumb Clover Trifolium 250–300 Trifolieae Fabaceae Rhizobium leguminosarum bv. trifolii Medic/ Medicago 50–100 Trifolieae Fabaceae Sinorhizobium meliloti melilot Melilotus 20 Trifolieae Fabaceae S. meliloti Trigonella 70–75 Trifolieae Fabaceae S. meliloti Pea Pisum 6ViciaeFabaceaeR. leguminosarum bv. viciae Lathyrus 130 Viciae Fabaceae R. leguminosarum bv. viciae Lens 5ViciaeFabaceaeR. leguminosarum bv. viciae Vicia 150 Viciae Fabaceae R. leguminosarum bv. viciae (Vavilovia) (1) Viciae Fabaceae R. leguminosarum bv. viciae Bean Phaseolus 50–100 Phaseoleae Fabaceae R. leguminosarum bv. phaseoli R. etli R. tropici Lupin Lupinus 150 Genisteae Fabaceae Bradyrhizobium sp. (Lupinus) Ornithopus 15 Hedysareae Fabaceae Bradyrhizobium sp. (Lupinus) Soybean Glycinec 2c Phaseoleae Fabaceae B. japonicum Cowpead All others ca 19,000 Very many Fabaceae Many species; fast- and slow-growers tribes

Mimosaceae Many species; fast- and slow-growers Caesalpiniaceae Several species; mainly slow-growers Source: derived from Fred et al. (1932). a According to Allen and Allen (1981). b According to Young (1996). c Glycine max and G. soja only; other species of the genus Soja allocated to the cowpea group. d Species of the genus Acacia allocated to the cowpea group.

62 nitrogen fixation in acacias

During the past decade, the concept of cross requirement for N fi xation, clovers from the Rocky nodulation has been modifi ed by advances in Mountains in North America a fourth, South rhizobial taxonomy (e.g. Young 1996; Young and American clovers a fi fth, and so on (Brockwell 1998). Haukka 1996). A consequence is that rhizobial It seems likely that these diff erent requirements nomenclature is no longer determined by the host for eff ective strains of R. leguminosarum bv. range of the organism, but by molecular techniques. trifolii are a consequence of the diff erent clusters Th e host range of the organism is then determined of Trifolium species having evolved in isolation quite independently. Despite this change of from one another. It represents a fi ne example of emphasis, the modern version of phylogenetic host/rhizobial specifi city. In contrast, acacias from relationships in the Rhizobiaceae (Table 3) still Australia, Africa and Asia are, more often than has substantial relevance to cross nodulation and not, nodulated by each other’s rhizobia. Th is is an retains implications for the selection of strains example of host/rhizobial promiscuity. for use in legume inoculants. Table 3 shows that (fast-growing) isolates of rhizobia from Acacia Th e concepts of specifi city and promiscuity belong to Sinorhizobium saheli. Some symbiotic have important practical implications for the affi nity between Acacia rhizobia (Sinorhizobium manipulation of the symbiosis between legumes saheli) and species of Leucaena, which is normally and rhizobia in order to enhance nodulation and nodulated by strains of the genus Rhizobium, has improve N fi xation. It has been known for more been demonstrated by Swelin et al. (1997). Th ere than a century that not all legumes are nodulated by are many similar anomalies. Undoubtedly, other all rhizobia. Th e extent to which a rhizobial strain (slow-growing) acacia rhizobia are unnamed species can infect and form nodules on diff erent legumes of Bradyrhizobium. Very-slow-growing isolates may is an inverse measure of its specifi city. Th e more have affi nities to Bradyrhizobium liaoningense. legumes it can nodulate the less specifi c, or more promiscuous, it is. A strain that has a very narrow Th e term ‘cross nodulation’ simply implies that a host range is considered to be highly specifi c. rhizobial strain has the ability to form nodules on the Identical terminology is used for the host plant. legumes in its cross-nodulation group. It does not A legume that accepts infections from and nodulates necessarily mean that those nodules will fi x nitrogen. with only a small number of rhizobial strains is Th e genus Trifolium provides examples of this termed specifi c; one that nodulates with many is constraint. In general, clovers of the Mediterranean promiscuous. Th e concept of specifi city applies to region fi x N with one particular set of strains of R. N fi xation as well as to nodulation. Combinations leguminosarum bv. trifolii, whereas clovers of central of plant and bacterium that form nodules do not African origin require a distinctly diff erent set of always fi x N. A consequence of this is that legumes strains for N fi xation. Clovers from the Caucasus and rhizobial strains are, as a rule, more specifi c for Mountains (north-eastern Turkey, southern Russia N fi xation than for nodulation. and Georgia) form a third cluster of rhizobial

63 nitrogen fixation in acacias

On the basis of ability to form nodules, many Acacia the soil to compensate for symbiotic ineffi ciency is species can be classed as promiscuous whereas, a trait that it apparently shares with other legumes, based on ability to fi x N, some of those same species e.g. Medicago polymorpha (common burr medic) are clearly specifi c. An example is A. caven (Frioni et (Bowman et al. 1998). al. 1998a,b). Working with a collection of strains of Bradyrhizobium, Turk and Keyser (1992) concluded We note that cross-nodulation occurs right across that A. mangium was promiscuous for nodulation the three families of legumes. Th is implies that but specifi c for N fi xation, whereas A. auriculiformis the root-nodule bacteria (rhizobia) from plants of was promiscuous for both nodulation and N one family have the capacity to form nodules and, fi xation. Woldemeskel and Sinclair (1998) went perhaps, to fi x N with some members of each of further and identifi ed rhizobial strain specifi city the other two families. It is likely, nevertheless, at subspecies and provenance levels in A. nilotica. that the symbiotic associations between some Burdon et al. (1999) went further still and drew acacias and their rhizobia are quite specifi c, with attention to the occurrence of specifi city in N little or no cross-nodulation with other species. fi xation between individual acacia seedlings. On the other hand, rhizobia isolated from acacia In Senegal, diff erent provenances of A. albida, nodules may have the ability to nodulate distantly approximately equal in total dry matter and N related legumes. For instance, Swelin et al. (1997) content, diff ered widely (0–38% Ndfa) in the noted the sparse nodulation of leucaena (Leucaena proportion of plant N due to N fi xation (Gueye et leucocephala) by strains of Bradyrhizobium sp. al. 1997). Th e ability of A. albida to scavenge N from (Acacia). Alison Jeavons Alison Jeavons Kennedia prostrata (running postman) — family Th e creeping shrub, Hardenbergia violacea (false Fabaceae — cross-nodulates with Acacia spp. An Australian sarsaparilla) — family Fabaceae — is another Australian native native, it is sometimes used as an ornamental ground cover. plant that cross-nodulates with Acacia spp. Hardenbergia violacea is a popular ornamental.

64 nitrogen fixation in acacias

Lorquin et al. (1997) determined the structures of other group exhibited reverse reactions. Ndiaye nod factors produced by strains of Sinorhizobium and Ganry (1997) recognised a consequence of teranga bv. acaciae and Rhizobium loti U cluster — promiscuity. When they detected low levels of almost certainly synonomous with what is now N fi xation in A. albida growing in the fi eld in known as Mesorhizobium plurifarium (de Lajudie et Senegal, they attributed it to paucity or absence al. 1998b) — both of which nodulate Acacia species. of appropriate rhizobia in the soil. Trinick (1980) Compounds from the two organisms were similar, located fast-growing strains that cross-nodulated indicating a close relationship between nod-factor with A. farnesiana and species of Lablab, Leucaena, structure and host specifi city, independent of the Mimosa and Sesbania. Sanginga et al. (1989a) taxonomic classifi cation of the rhizobia. reported that some slow-growing strains — of Bradyrhizobium sp. (Faidherbia) — isolated from A. albida could form nodules on leucaena (Leucaena Rhizobial specifi city and promiscuity leucocephala). Rasanen et al. (2001) noted that As might be expected in so large and diverse a genus, strains of four species of Sinorhizobium isolated in there are all degrees of host/rhizobial specifi city in Sudan and Senegal nodulated and fi xed N with eight the Acacia symbiosis, varying from highly specifi c to Acacia species and four Prosopis species of African widely promiscuous (e.g. Habish and Khairi 1970; or Latin American origin, but not with Sesbania Roughley 1986; Bowen et al. 1999). Indeed, some rostrata. Lopez-Lara et al. (1993, 1995) noted that extraordinarily promiscuous rhizobia, e.g. Rhizobium a strain of Rhizobium sp. from A. cyanophylla had sp. strain NGR234 isolated from the non-legume a very wide host range that included Trifolium Parasponia andersonii, have a diverse host range of species. Th ey implicated the composition of surface 300–400 species including acacias (Pueppke and polysaccharides in the promiscuity. Wang and Wang Broughton 1999). Th e literature holds a number (1994), on the other hand, reported that a strain of of examples of specifi city and promiscuity relating rhizobia similar to Sinorhizobium meliloti, which had to both nodulation and N fi xation by acacias. Njiti been isolated from A. auriculiformis, nodulated its and Galiana (1996) classifi ed the tropical, dry- host and A. confusa at high frequency, A. mangium zone species, A. albida, A. holosericea, A. nilotica, A. at low frequency, but formed no nodules at all on polyacantha and A. senegal, as promiscuous in that A. mearnsii. Only one of 12 strains of acacia rhizobia they all nodulated and fi xed N indiscriminately with isolated from A. mangium in the Philippines formed strains of both Rhizobium and Bradyrhizobium. In eff ective nodules on its host (Dart et al. 1991). contrast, Dommergues (1982) demonstrated more specifi c reactions, as interactions between two Th ompson et al. (1984) took 38 strains of rhizobia groups of acacia hosts and diverse rhizobia. One isolated from Australian native legumes, including group fi xed N with fast-growing strains (Rhizobium, acacias, and used them in a glasshouse experiment and probably Sinorhizobium and Mesorhizobium) to inoculate 63 species of Acacia. Th e majority of but not with slow growers (Bradyrhizobium); the species (45/63) formed nodules with more than

65 nitrogen fixation in acacias

75% of the rhizobial strains, 11/63 nodulated with and micro-organism might be exploited to enhance between 50% and 75% of strains and 7/63 with less the symbiosis. Th ey found evidence that N fi xation than 50%. No data were presented for N fi xation. might be increased by selecting both the lines of Dart et al. (1991) demonstrated a wide range of A. mangium and the rhizobia that nodulate it. specifi city and symbiotic eff ectiveness when they tested 48 strains of acacia rhizobia of diverse Appraisal of specifi city as a constraint to origin on A. auriculiformis and A. mangium. Acacia eff ective fi eld nodulation of acacias auriculiformis was very much more promiscuous in terms of N fi xation than A. mangium. Th ey used While it is clear that Acacia host/Acacia rhizobia these and related fi ndings as a basis for selecting symbioses are extremely variable, we conclude N-fi xing rhizobia for particular acacia species that most associations between the symbionts in (Bowen et al. 1999). Burdon et al. (1999) describe a the wild usually fi x some atmospheric N. Although remarkable instance of Acacia host/Acacia rhizobia the amount fi xed varies considerably, we believe specifi city in A. dealbata. Ten half-sib families were that the symbiosis itself is unlikely to be the major each inoculated with a single strain of rhizobia. determinant of how much N is actually fi xed. In nine of the families, N fi xation was ineff ective, It seems to us that it is much more likely that whereas the tenth fi xed N vigorously. environmental factors, particularly the presence of soil N as NO₃–, but also soil reaction, soil moisture, soil salinity, nutrient defi ciency, insuffi cient light, Variation in the symbiotic reaction between and the infl uence of pests, limit the amount of N host and rhizobia fi xed by naturally occurring acacias. It is only when Th e degree of specifi city shown by a species of Acacia limiting factors such as these are not operating or a strain of rhizobium is undoubtedly a function of that the N-fi xing ability of a legume/rhizobium the particular combination of plant and bacterium association will be be capable of expressing its used to make the determination. Table 8 shows an full potential. Th en, and only then, might partial example taken from the work of Turk (1991), who eff ectiveness of the symbiosis be a factor limiting tested three Acacia species against a diverse collection N fi xation. Th ese arguments would not, of course, of strains from three genera of rhizobia. Th e apply to those cases when the symbiotic association responses in terms of nodule formation are complex, between plant and bacteria is ineff ective. However, but it is clear from the data that the complexities we suspect that the occurrence of truly ineff ective of the interactions would be diff erent had a smaller associations in the fi eld is very rare. An appraisal of subset of the strains, or another collection of strains, the literature that we have cited supports this view. been used for making the determination. A form of selective preference for particular Souvannavong and Galiana (1991) presented an bacteria for nodule formation has been recorded example of how the interaction between legume for Acacia species by Odee et al. (1998). Th ey found

66 nitrogen fixation in acacias that, when grown in various African soils, each Table 8. Nodulation of three species of Acacia containing a diverse rhizobial microfl ora, A. albida, inoculated with 34 diverse strains A. auriculiformis and A. holosericea nodulated largely representing three genera of root- with Bradyrhizobium and only occasionally with nodule bacteria Rhizobium, whereas A. polyacantha and A. tortilis grown in the same soils nodulated exclusively with Rhizobium. Certain legumes (e.g. Trifolium spp.) exercise a more specifi c form of selective preference Species by selecting for their nodulation the more eff ective strains from a mixed rhizobial microfl ora — see e.g. Robinson (1969) and Masterson and Sherwood of strains No. A. auriculiformis mangium A. A. mearnsii Rhizobium 18 (1974). Th ere is some unconfi rmed evidence 2 Plusa Plus Plus that this phenomenon might also occur in Acacia 2 Plus Plus Minusa (Table 9). 2 Plus Minus Minus 3 Minus Plus Plus Nevertheless, even in circumstances where the 2 Minus Plus Minus N-fi xing capacity of fi eld populations of acacia 7b Minus Minus Minus rhizobia is defi cient, it would seem ecologically Bradyrhizobium 15 and economically impossible to introduce new, 6c Plus Plus Plus more eff ective strains into the soils of established 1 Plus Plus Minus forests. Enhancement of the amount of N fi xed by 3 Plus Minus Plus forest acacias might be feasible by correction of 1 Minus Plus Plus soil nutrient defi ciencies but even that is probably 1 Minus Plus Minus impracticable. Notwithstanding, we believe 2 Minus Minus Plus that there is substantial scope for exploiting the 1 Minus Minus Minus symbiosis to improve the health and vigour of Azorhizobium 1 acacias newly established in plantations, for farm 1 Plus Plus Plus forestry or in land rehabilitation (e.g. Th rall et al. 2001a,b). Source: data extracted from Turk (1991). a Plus = nodules; Minus = no nodules. b Th ree strains from Robinia, not classifi ed by Turk (1991), thought to be Rhizobium. c One reaction, not determined, thought to be positive (Plus).

67 nitrogen fixation in acacias

Table 9. Evidence for selective preference* by Acacia species for the more eff ective rhizobial strain components of a mixed-strain inocula

(a) Eff ectiveness index for three strains of acacia rhizobia in association with three Acacia species. (Index is the whole plant dry matter increment due to inoculation) Strain of rhizobia A. implexa A. melanoxylon A. mearnsii 53A-21 0.90a 1.25ab† 1.18a 49A-20 0.84a 1.55a 1.26a 4207 0.71a 0.79b 0.94a

(b) Responses (dry matter — mg/plant) of three Acacia species to inoculation with three single-strain inocula and a multi-strain inoculum Strain of rhizobia A. implexa A. melanoxylon A. mearnsii Single-strain inocula 53A-21 415ab† 301ab 358a 49A-20 384ab 351a 367a 4207 367b 238c 312a Multi-strain inoculum All three 443a 341ab 325a

* Selective preference is indicated when the response to inoculation with a mixed-strain inoculum (of 2 or 3 strains) is equal to or greater than the response to inoculation with any one of the strains as a single-strain inoculum. For instance, strain 4207 has a relatively poor eff ectiveness index (see part a) with each of the three Acacia species. When 4207 is used as a single-strain inoculum, its N-fi xing performance (with each of the three species) is inferior to the performance of multi-strain inocula of which 4207 is a component (see part b); i.e. the Acacia species have exercised a selective preference for the better strains in the mixed-strain inocula. † Values in each row with a common letter are not signifi cantly diff erent from one another (P>0.05).

68 5. Enhancement of the symbiosis

the need for inoculation the likely success of introducing inoculant rhizobia Acacia rhizobia are, as we have mentioned, as into the soil by considering indices of the size of the widely distributed as the Acacia species themselves. resident rhizobial population and the N status of Indeed, they sometimes occur spontaneously in the soil (cf. Singleton and Tavares 1986). Th ies et al. anthropogenic environments, e.g. landfi ll in Hong (1994) put forward a unique proposal for predicting Kong (Chan et al. 1998). Nonetheless, there are the need for inoculation on a regional basis using a many soils where the population density is so low geographical information system. as to pose a threat to the establishment of N-fi xing trees (Th rall et al. 2001a). Th ere will be other soils As our literature survey has shown, there are, among where suitable strains for rhizobia-specifi c species acacias, all degrees of host/rhizobial specifi city in will be absent. Used judiciously according to need, the symbiosis, varying from widely promiscuous to and performed properly, legume inoculation is a highly specifi c. Species in the latter category are those signifi cant agency for improving plant productivity most likely to need and benefi t from inoculation and soil fertility. ‘Is it necessary to inoculate?’ is a upon introduction into new environments where question that has been approached in diff erent ways soils lack specifi c N-fi xing rhizobia. (Table 10). responses to rhizobial Field experiments designed to diagnose the need for inoculation of acacias in field inoculation (e.g. Brockwell 1971; Date 1977; Th ies et and nursery al. 1991a) are unsuitable for acacias. Bonish (1979) and Brockwell et al. (1988) used dilutions of soil Masutha et al. (1997) suggested that promiscuous samples to inoculate clover seedlings growing under species of Acacia, capable of nodulation and N aseptic conditions in test tubes to demonstrate a fi xation with naturalised populations of rhizobia, quick, microbiological means for characterising should be chosen for use in agroforestry programs, simultaneously the size and N-fi xing capacity of soil- implying that they considered natural inoculation borne populations of rhizobia. Th e method could, of acacias by resident soil rhizobia to be superior no doubt, be refi ned for use as a diagnostic of the to artifi cial inoculation with cultures of rhizobia. need for inoculation of acacias. A related procedure We accept that proposition in situations where the (Th ies et al. 1991b) makes it possible to forecast numbers of acacia rhizobia in the soil are so large

69 nitrogen fixation in acacias

(say >1000 cells per gram) that introduced strains 42 months after transfer of the inoculated trees to would encounter extreme competition from the the fi eld. Th is particular organism (Aust13c) appears resident organisms. Where, however, naturalised to have considerable potential as a strain for making populations are smaller, there are excellent inoculants. In Ivory Coast, 50–90% of total nitrogen prospects of successfully introducing inoculant in A. mangium trees was attributed to fi xation strains, provided sensible strategies are employed. of atmospheric N (Galiana et al. 1996). Galiana et al. (1998) reviewed these works and similarly While there are only a few fi eld reports of acacias successful experiments with A. mangium that had responding to rhizobial inoculation, they document been conducted in other tropical countries. Lal and careful work and convincing results. Working with Khanna (1996) demonstrated a fi eld response to A. mangium in Ivory Coast, Galiana et al. (1994) inoculation of A. nilotica grown in India. reported that inoculation had a positive eff ect on tree growth for more than three years after Th e long-term success of inoculation in the fi eld outplanting. Moreover, one of the inocula persisted appears to depend on the initial establishment well and could be re-isolated from root nodules up to of a vigorous crop of nodules. Th e nodule itself

Table 10. Indicators of the need to inoculate legume seed with eff ective rhizobia at time of sowing

Allen and Allen (1961) — historical indicators 1. Th e absence of the same or of a symbiotically related legume in the immediate past history of the land 2. Poor nodulation when the same species was grown on the land previously 3. When a legume follows a non-legume in a rotation 4. In land reclamation undertakings.

Roughley and Brockwell (1987) — microbiological queries 1. How specifi c is the legume in its rhizobial requirements? 2. What is the likelihood of eff ective rhizobia spreading from volunteer legumes? 3. Has the legume been sown previously and for how long was it grown continuously? 4. How long since the legume was last grown and, in the meantime, did conditions favour persistence of the rhizobia?

Th ies et al. (1991b) — queries relating to soil indices 1. How large is the resident population of competitive rhizobia? 2. What is the level of soil nitrogen? Source: after Brockwell et al. (1995a).

70 nitrogen fixation in acacias represents an environment akin to pure culture tube stock. Th is practice can be exploited for and, within it, there is great multiplication of the inoculation. Acacias can be inoculated at the time rhizobia. When, subsequently, nodule breakdown of sowing in the nursery in a way that ensures that takes place, large numbers of viable cells are the seedling trees are vigorous, well-nodulated and released into the soil (e.g. Reyes and Schmidt fi xing N at the time of outplanting into the fi eld. 1979; Kuykendall et al. 1982; Moawad et al. 1984; Th is has been demonstrated at nurseries in Australia Th ies et al. 1995) where they constitute a potent (Brockwell et al. 1999a). When nursery-inoculated source of infection for new roots and may become a seedlings were outplanted, their survival, growth permanent component of the soil microfl ora even in and benefi t to companion plantings of Eucalyptus the presence of competing organisms. nitens (shining gum) was better than that of uninoculated seedlings (Table 11). Th e total area of land worldwide under acacia plantations exceeds 5 million hectares. It is probable Wherever restoration of native vegetation and that most of these trees were outplanted as nursery re-establishment of tree cover over large areas

Table 11. Eff ect of inoculation by soil enrichment on growth of Acacia mearnsii in nursery and fi eld. (N fi xed calculated using natural abundance of 15N with Eucalyptus nitens as the non-N-fi xing reference plant)

A. mearnsii LSD (P=0.05) Inoculated Uninoculated (n.s. — P>0.05) Nursery Seedlings nodulated (%) — day 126 100.0 35.0 33.0 Seedlings nodulated (%) — day 231 100.0 70.0 n.s. Nodule score (0-5) — day 126 4.00 0.60 0.82 Nodule score (0-5) — day 231 3.00 0.95 0.73 Shoot DM (g/plant) — day 231 1.78 1.27 n.s. Shoot DM (mg/plant) — day 231 35.7 26.4 14.6 Shoot N fi xed (%) — day 126 68.3 44.4 5.9 Shoot N fi xed (%) — day 231 88.3 25.3 11.8 Shoot N fi xeda (mg/plant) — day 126 31.6 7.8 12.3 Fielda Survival (%) — day 231 to day 280 96.3 90.7 4.6 Survival (%) — day 280 to day 553 98.1 91.4 3.4 Tree height (cm) — day 280 28.3 21.2 1.2 Tree height (m) — day 553 2.03 1.82 0.9 Source: after Brockwell et al. (1999a). a Seedling trees outplanted into the fi eld after 231 days in the nursery.

71 nitrogen fixation in acacias are major conservation issues, as in some parts who produces (except by special request) rhizobial of Australia, the use of tube stock becomes cultures for Acacia species. Until that situation is impracticable. Where acacias (or other leguminous rectifi ed, this important natural, renewable resource trees or shrubs) are appropriate for such will remain under-exploited. Inoculant preparation circumstances, it may be a more practical sowing involves only a few, simple, well-established steps. strategy to use direct drilling or aerial seeding (Th rall et al. 2001a,b). As part of the methodology, inoculants it would be necessary to incorporate rhizobial inoculant in a seed pellet (e.g. Brockwell 1962) Th e principles of inoculant production are well or to deliver the inoculant in granular form (e.g. documented — e.g. Burton (1982); Th ompson Scudder 1975) into the seed furrow close to the (1983); Somasegaran and Hoben (1994); other work seed. It is suggested that direct drilling or aerial cited by Brockwell et al. (1995a). Th e selection of seeding approaches using multi-strain inocula carrier material is a critical determinant of inoculant (see below) incorporated in seed pellets should quality. Finely ground peat (Th ompson 1980) has be adopted as a general strategy for revegetation been the inoculant carrier of popular choice for of degraded landscapes with shrubby legumes in many years, though there are other promising Australia and, perhaps, other parts of the world carriers (Brockwell and Bottomley 1995). Roughley where native forest has been removed. Used in and Vincent (1967) and Date and Roughley (1977) conjunction with the replanting of Eucalyptus and/ record that inoculants prepared with sterile peat or other non-leguminous species, the benefi ts are contain 100-fold more rhizobia than those made likely to be enhanced. Overall, the development of with non-sterile peat. Moreover, because mortality practical approaches and uncomplicated techniques of rhizobia is greater in unsterilised peat, the suitable for large-scale, low-cost establishment of diff erence increases during storage. native legumes is likely to lead to more effi cient revegetation and soil management by landholders, Strain selection and greater incentive to invest in reclamation projects due to aff ordable costs and higher survival Some principles of matching acacia species and rates of the sown species. acacia rhizobia have been catalogued by Brockwell (1998). A list of characters considered desirable for Th e paucity of literature relating to fi eld inoculation rhizobial strains for legume inoculants is shown in of acacias indicates that most plantings rely Table 12. It is obviously not feasible to test strains on spontaneous nodulation from naturalised for all of these characters. Naturally, the ability to populations of rhizobia. Clearly, acacia symbioses form nodules and fi x N on the target legume are with eff ective rhizobia are not properly exploited. the essential characteristics. Methods for testing Indeed, at the time of writing, we are unaware of for strain eff ectiveness for legumes in general any inoculant manufacturer anywhere in the world have been described by, amongst others, Vincent

72 nitrogen fixation in acacias

(1970), Gibson (1980) and Somasegaran and Hoben Table 12. Characters considered desirable for (1994). Methods adapted or specifi cally designed for inoculant strains and inoculant carriers acacias have been used by Th ompson et al. (1984), Umali-Garcia et al. (1988), Galiana et al. (1990) and Strain characters for legume inoculants Burdon et al. (1999). Using such methods, Brockwell 1. Ability to form nodules and fi x N on the target et al. (1999b) and Dippel et al. (1999) made legume recommendations for inoculant strains for a range of 2. A wide host range, i.e. the ability to fi x N with a acacias to be raised in plant nurseries and destined wide range of host genotypes for plantation and farm forestry. Martin-Laurent et 3. Ability to fi x N across a wide range of al. (1997) demonstrated that aeroponic culture led environmental conditions to abundant nodulation of inoculated A. mangium. 4. Ability to compete in nodule formation with populations of rhizobia already present in the soil Th e method lends itself to studies of infection 5. Ability to form nodules and fi x N in the presence of processes and nodule morphology and, if it can be soil nitrate adapted, to selection of eff ective rhizobial strains. 6. Ability to grow well in artifi cial media, in inoculant carrier and in the soil As noted earlier, there are all degrees of host/ 7. Low mortality on inoculated seed rhizobial specifi city in the acacia symbiosis. 8. Ability to migrate from the initial site of However, a pragmatic approach to strain selection is inoculation essential for production of acacia inoculants because 9. Ability to tolerate environmental stress it would not be feasible to have a special inoculant 10. Ability to colonise the rhizosphere of the host for every diff erent acacia. With respect to the plant N-fi xing potential of any particular acacia/inoculant 11. Ability to colonise the soil in the absence of a legume host strain combination, we advocate the principle of 12. Genetic stability seeking the best result possible rather than the best 13. Compatibility with agrichemicals possible result. Th e fi ndings of Burdon et al. (1999) and Th rall et al. (2000) give some encouragement. In Properties of good inoculant carriers studies of symbiotic associations between temperate Australian acacias and populations of native 1. High water-holding capacity rhizobia, they found a general lack of host/rhizobia 2. Non-toxic to rhizobia interaction eff ects (with notable exceptions) and 3. Easy to sterilise by autoclaving or gamma irradiation concluded that, where no rhizobial strain for a 4. Readily and inexpensively available particular host species is available, strains from its 5. Suffi ciently adhesive for eff ective application to seed closest relative will have the highest probability of 6. Good pH buff ering capacity success in N fi xation. 7. Good cation- and anion-exchange capacities Source: Th ompson (1980), Keyser et al. (1992), Brockwell and Bottomley (1995), G. Bullard (pers. comm.)

73 nitrogen fixation in acacias

Th ere appears to be scope for selecting strains that When commercial manufacture of legume are adapted to harsh conditions. For instance, inoculants began in the 1890s, liquid (broth) culture Surange et al. (1997) identifi ed acacia rhizobia that was the preferred form. Nowadays, the vast majority were tolerant of high salt concentrations and high of inoculants are prepared in powdered organic alkalinity, and Kang et al. (1998) acid-tolerant strains. carriers such as fi nely ground peat. Nevertheless, it is feasible to manufacture and market high-quality liquid inoculant (Lie et al. 1992; Brockwell et al. Inoculant production 1995a). Indeed, in their experiments described Th ere is a profuse literature on all aspects of large- earlier, Galiana et al. (1994, 1996, 1998) used liquid and small-scale preparation of legume inoculant. inoculum for nursery-grown acacia seedlings that Th e major papers on the principles and practice were then outplanted. Existing inoculants of highest include Vincent (1970), Burton (1976, 1979, quality tend to be those produced by small factories 1982), Brockwell (1977), Date and Roughley under the umbrella of a quality control authority, (1977), Th ompson (1980, 1983), Williams (1984), such as those described by Roughley and Pulsford Somasegaran (1985, 1991), Diem et al. (1989), (1982), Th ompson (1983) and Somasegaran (1991). Keyser et al. (1992), Smith (1992) and Somasegaran Th e NifTAL Center and MIRCEN in Hawaii have and Hoben (1994). developed a ‘micro-production unit’ for small-scale production of rhizobial inoculants (NifTAL Center Th e fi rst step in inoculant preparation is the 1993). Th ere are two key concepts: (i) use of a production of a broth with, desirably, a population sterile peat carrier (cf. Date and Roughley 1977) density of at least one billion (1 × 10⁹) viable rhizobial and (ii) dilution of the rhizobial broth culture with cells per mL. Th e broth is grown in a fermenter which sterile water (Somasegaran and Halliday 1982; can be a very simple piece of equipment (Date and Somasegaran 1985) immediately before it is injected Roughley 1977). Th e broth is then incorporated into the sealed bag of peat carrier. Th e unit has a in fi nely ground peat which preferably has been number of advantages over conventional (larger- sterilised by gamma-irradiation (Roughley and scale) methods for producing legume inoculants. Vincent 1967) or autoclaving. Finally, the peat First, the unit is cheap and easy to assemble. culture is packaged and sealed into polyethylene bags Second, the number and size of fermentors used of convenient size, ‘matured’ at 25°C for 14 days for propagating broth cultures of the rhizobia are and stored, usually at about 4°C, until it is needed. reduced by using the dilution technique. Th ird, Th e demand for acacia inoculant is likely to be met production schedules are very fl exible. Fourth, by small fermenters. Balatti (1982), Hoben and because the unit uses sterile peat as a carrier, Somasegaran (1992) and Somasegaran et al. (1992) populations of rhizobia in the inoculant may be provide specifi cations for small fermenters, any of 10 times greater than in the many commercial which would be suitable for the production of high- inoculants manufactured from non-sterile carriers titre broths of strains of acacia rhizobia. (cf. Roughley and Vincent 1967; Brockwell 2004).

74 nitrogen fixation in acacias

Fifth, the use of sterile peat increases shelf-life over rhizobia are very much obligated to their hosts. that of products made with non-sterile carriers. Virtually all rhizobial multiplication in the wild Sixth, the unit requires little space. Last, quality- takes place in the host rhizospheres, on the root control procedures are simple and reliable. NifTAL’s surfaces and especially within the nodules. When micro-production unit appears ideal for preparing an inoculant culture is applied to the seed surface the relatively small quantities needed to meet the or introduced into a seed bed or a nursery soil tube, likely demand for acacia inoculants. the rhizobia start to die. Th e mortality continues until the development of a seedling rhizosphere that can be colonised by the surviving rhizobia. Initial Multi-strain inoculants procedures should therefore aim (i) to maximise Th e current practice in Australia is to use a single survival of the inoculant during the non-rhizosphere eff ective strain of rhizobia in commercial legume period, and/or (ii) to accelerate germination so that inoculants (Roughley et al. 1984). Species-specifi c the non-rhizosphere period is reduced. Figure 3 strains have been advocated for use in inoculants illustrates the principles involved. for certain Acacia species, e.g. A. mearnsii (Turk and Keyser 1992). Th e use of single-strain inoculants For inoculation of acacias, the principles are easily facilitates quality control (Th ompson 1980), but implemented. Currently, most acacia plantings in has the major disadvantage of restricting the the fi eld are made using nursery stock raised in scope (host range) of any particular inoculant. It is tubes of soil. Inoculant can be incorporated into the common practice in most other countries to produce nursery soil immediately before planting the acacia multi-strain inoculants. Somasegaran and Bohlool seed. Th is general procedure was described in each (1990) made an extensive comparison of multi- and of three reports of fi eld benefi ts of inoculation: Lal single-strain inoculants. Th ey found that, in almost and Khanna (1996) used seed inoculation, Galiana all cases, the N-fi xing eff ectiveness of a multi-strain et al. (1994) applied liquid inoculum to the nursery inoculant exceeded or equalled the performance soil, and Brockwell et al. (1999a) mixed peat culture of the best strain in that inoculant. We therefore into the soil. We believe that the last procedure, consider that use of multi-strain inoculants is termed inoculation by soil enrichment, best meets essential to achieve successful nodulation and N the demands of the principles illustrated in Figure fi xation in acacias whenever it is proposed to use the 3. Higher rates of inoculation can be achieved by one inoculant for more than one species. incorporating an inoculant into the soil than by applying it to the seed surface, and experience has shown that peat-based inocula tend to survive Inoculation strategy better in soil and in the fi eld than do liquid inocula Although classifi ed as free-living organisms with (e.g. Brockwell 1962). saprophytic properties (e.g. Chatel et al. 1968),

75 nitrogen fixation in acacias

Indeed, some operators actually boil their acacia seed for one minute. (Strictly speaking, with most acacia seed, the pre-treatment does more than merely accelerate germination; it triggers processes that stimulate germination.) Naturally, rhizobial enrichment of the soil should be done immediately before planting the seed. To avoid death of the inoculant from desiccation and/or heat stress, the soil should, after planting, be kept moist and as cool as practicable by the use of a misting device. It is pertinent that Gassama-Dia (1997), using delayed inoculation experiments, demonstrated that the period of maximum infectibility of A. albida inoculated with Bradyrhizobium was the 13 days immediately after seed germination.

A particular advantage of soil enrichment inoculation is that it lends itself to the preparation and use of inoculant strains selected for special purposes, e.g. tolerance of salinity, acidity, alkalinity and other forms of environmental stress.

Th ere will be situations, as in large-scale land Figure 3. A schematic illustration of basic factors rehabilitation (cf. Th rall et al. 2001a,b), prevention involved in improving the likelihood of of dryland salinisation and generation of carbon, legume nodulation following rhizobial conservation and biodiversity credits, when it is inoculation: (i) reducing inoculant desirable to establish acacias by direct seeding. mortality, (ii) increasing rate of inoculant Alternative means of inoculation would be required application, (iii) decreasing time to in such circumstances. In studies of rhizobial germination (after Brockwell 1962). survival following aerial seeding, Hely (1965) demonstrated that inoculation and seed coating Accelerated germination of acacia seed can be produced vigorous swards of well-nodulated, eff ected by a pre-treatment of immersing the N-fi xing crimson clover (Trifolium incarnatum). It seeds in near-boiling water for a short time and might be possible to develop a similar procedure for then allowing them to imbibe the water as it cools. acacias.

76 nitrogen fixation in acacias breeding and selection for to perennial tree and shrub species such as acacias. enhanced acacia symbiosis On the other hand, the simple, long-standing Because acacias are essentially non-domesticated techniques for selecting for improved symbiosis in plants, there must be some scope for breeding and poorly domesticated forage plants that have been selecting certain species for increased capacity described by Hutton and Coote (1972) for greenleaf to nodulate and fi x N. We do not know, however, desmodium (Desmodium intortum) and by Zorin et whether the sort of classical procedures outlined al. (1976) for Caucasian clover (Trifolium ambiguum) by Sprent (1994c) for an annual crop plant such as might have application to acacias. groundnut (Arachis hypogaea) would be applicable Alison Jeavons Acacia genistifolia

77 6. Exploitation of the symbiosis

We have already pointed out that acacias are quite large A. albida trees, and to a lesser extent A. tortilis, capable of existing in the absence of an eff ective in cultivated fi elds. Because the trees are deciduous symbiosis. Th ere is no pretending that the quality in the rainy season, they do not compete for light of some acacia products, such as perfume oils and with crop plants grown beneath them (cf. Giller and oyster poles, is infl uenced in any way by N fi xation. Wilson 1991). Also, A. albida may add substantial However, sometimes it is diffi cult to separate N to the soil as a result of the leaf fall that occurs cause and eff ect. In Australia, A. harpophylla is at about the time that cash crops are sown. Indeed, almost always associated with soils of high N in the Sahel, continuous dry-season cropping with status (Graham et al. 1981), but it is not possible sorghums and millets has been practised beneath to know with certainty whether the soil is high in A. albida without reductions in yields or additions N due to the presence of the acacia or if the acacia of fertilisers (Porteres 1954). Yields of groundnut needs fertile soil to grow. Notwithstanding, there (Arachis hypogea) and cereals are often much higher is no doubt that there are also many circumstances under A. albida trees than in open fi elds (e.g. where the N fi xed by acacias represents a valuable Charreau and Vidal 1965; Radwanski and Wickens commodity. 1967; Dancette and Poulain 1969; Charreau and Nicou 1971; Felker 1978; Poschen 1986). Moreover, the litter of A. albida and A. tortilis improves the soil natural exploitation environment by contributing to retention of soil African peoples have long recognised that the moisture through an increase in soil organic matter, extensively distributed, widely adapted indigenous improving soil structure, enhancing populations leguminous tree, A. albida, confers special benefi ts of soil microfauna, and reducing extremes of on its immediate environment and, therefore, has evapotranspiration and soil temperature (e.g a signifi cant place in agricultural practice (Felker Dancette and Poulain 1969; Bernhard-Reversat 1978; Giller and Wilson 1991; Saka et al. 1994). 1982; Young 1989). In addition to the N added Acacia tortilis has similar status in the dry savannas to the soil as a result of leaf and pod fall, there of East Africa (Giller and Wilson 1991). Th ese trees may well be further supplementation of soil N grow in the dry season and shed their leaves in the through underground release of N in disintegrating, rainy season. A traditional farming practice in many decaying roots and nodules. Much of all this is a African countries is the maintenance of parklands of consequence of the ability of A. albida and A. tortilis

78 nitrogen fixation in acacias to fi x atmospheric N (see Table 4) although the conditions for the re-establishment of other trees might also have the capacity to access deep- components of Hawaiian highland forests cleared soil N and N in groundwater. Additionally, both 150 years ago for pastoral use. Th e present rhizobial trees provide an abundance of fodder and fuelwood status of the soil, and therefore the need to throughout the year and deep shade in the dry inoculate the A. koa, is not known. season (e.g. Bunderson et al. 1990). Acacia albida may be the Earth’s most comprehensively utilised Acacias are used for rehabilitation of land damaged plant species and represents a classic example of by industrial waste (e.g. Zhang et al. 1998), mining natural exploitation of the legume symbiosis. activities (e.g. Langkamp et al. 1979; Franco and de Faria 1997) and landfi ll (e.g. Chan et al. 1998), and In both forest and savanna ecosystems, trees for other forms of land reclamation (e.g. Kirmse and substantially infl uence the chemical and physical Norton 1984; Assefa and Kleiner 1998) including characteristics of the soil system through a variety stabilisation of roadsides (e.g. Searle 1997). Th e of mechanisms, e.g. deposits of litter, the activity genus has great scope for reclaiming saline land of soil macro- and microfl ora involved in litter (Turnbull et al. 1998b). Coconut (Cucos nucifera) decomposition, and redistribution and accumulation responded to interplanting with A. auriculiformis of soil nutrients through the scavenging and by increasing its total root biomass (Arachchi and conduit properties of extensive root systems Liyanage 1998). Th ere is scope for using acacias to (Rhoades 1997). N-fi xing trees make an additional, revegetate and rehabilitate degraded landscapes, special contribution of N to the ecosystem and, such as the Imperata grasslands in Southeast Asia consequently, other things being equal, represent a (Turnbull et al. 1998b), the Chilean secano interior preferred form of vegetation in agroforestry. An apt (Arredondo et al. 1998), and the Atlantic lowlands example is the contribution made by Acacia species of Costa Rica (Tilki and Fisher 1998). Temperate and as natural understorey components of Eucalyptus tropical species of Acacia from Australia are suitable forest ecosystems in Australia (Adams and Attiwill for many of these purposes. 1984; Hansen and Pate 1987a,b). intercropping general exploitation Intercropping of a legume with a non-legume often Th ere are many examples of successful exploitation increases signifi cantly the amount of symbiotic of the genus Acacia, and its symbiosis with root- N fi xed by the legume and the total amount of N nodule bacteria. Th e endemic Hawaiian legume, uptake by the joint components of the system — A. koa, produces substantial biomass and fi xes e.g. rice bean (Vigna umbellata)/maize (Zea mays) abundant N (Pearson and Vitousek 1997). Scowcroft (Rerkasem et al. 1988); soybean (Glycine max)/ and Jeff rey (1999) considered that A. koa has maize (Martin et al. 1991); French bean (Phaseolus potential as a nurse crop to create understorey vulgaris)/maize (Pineda et al. 1994). It seems likely

79 nitrogen fixation in acacias that, at least partly, the uptake of soil N by the non- attributed to enhanced N status. A similar eff ect legume reduces the amount of soil N available to the has been obtained with A. mearnsii and Eucalyptus legume, thereby ‘forcing’ it to fi x more atmospheric nitens (shining gum) in south-eastern New South N in order to fulfi l its requirement for N. In some Wales (J. Brockwell, P.A. Mitchell and S.D. Searle, circumstances, however, intercropping might unpublished data). suppress legume yield when both plants compete for the same limited resources (e.g. Hakim et al. 1991). Khanna (1998) postulated that the improvement was brought about by underground transfer of N Intercropping has been successfully applied from the Acacia to the Eucalyptus. He ruled out to forestry. Turvey et al. (1983) reported that the possibility of above-ground transfer in his planted pine trees benefi tted from an association experiments because, at the time of increased with naturally regenerated Acacia species. Th e Eucalyptus growth, decomposition of litter on extent of the response was related to the density the soil surface was trivial and had contributed of the acacias. In experiments in plantations in little or nothing to the N dynamics of the system. Southeast Asia and Australia, Khanna (1997, Th e literature is equivocal on the question of 1998) grew mixed stands of fast-growing species underground transfer of legume N to associated of (tropical and temperate, respectively) Acacia and non-legumes. Th ere are arguments for — e.g. work Eucalyptus. Th e Acacia species were actively fi xing and citations by Ofi ri and Stern (1987); Viera- N. Th eir presence led to incremental growth in the Vargas et al. (1995) — and against — papers cited by Eucalyptus species (Table 13; Figure 4) which was Peoples and Craswell (1992).

Table 13. Mean tree basal area (cm2/tree) in pure and mixed stands of Eucalyptus globulus and Acacia mearnsii grown at two densities for 45 months at Cann River, Victoria, Australia

Tree density (ratio) Total tree density Eucalyptus Acacia 1000/ha 1500/ha Eucalyptus Acacia Eucalyptus Acacia 100 0 9.1 b* – 13.0 c – 75 25 11.4 ab 41.4 a 15.9 ab 49.1 a 50 50 14.1 a 29.3 b 18.2 a 42.5 b 25 75 14.3 a 24.3 cd 17.3 a 36.5 c 0 100 – 19.8 d – 25.2 d Source: after P.K. Khanna, unpublished data. * In any one column, values with a common letter are not signifi cantly diff erent from one another (P>0.05).

80 nitrogen fixation in acacias

Khanna’s (1997, 1998) evidence for underground improvement in the N nutrition of the eucalypts transfer of Acacia N to Eucalyptus is most was a consequence of association with acacias. convincing. In further studies involving fi ne-root architecture, researchers in Khanna’s laboratory It is interesting to speculate just how such transfers (Bauhus et al. 2000) found ‘high’ concentrations might occur. While trees are capable of using both of N in the fi ne roots of Eucalyptus globulus (blue nitrate and ammonium for growth (Devisser and gum) when it was grown in a 50:50 mixture with Keltjens 1993; Turnbull et al. 1995), it seems A. mearnsii. Th is was another indication that the unlikely that these products, released from the

Figure 4. Cartoon about the benefi ts of interplanting Eucalyptus and Acacia species (with acknowledgment to Th e Canberra Times)

81 nitrogen fixation in acacias organic N in root and nodule debris by classical case studies and, taken altogether, represent the nitrifi cation processes, would have been available conventional wisdom on the use of Acacia species in for underground transfer — for the same reasons land reclamation in southern Australia. that above-ground decomposition of litter was negligible. We favour a hypothesis centred on General principles mycorrhizal activity. Extensive, diverse populations of mycorrhiza are extremely common in forest Most undisturbed or lightly disturbed Australian ecosystems. Although Khanna (1997, 1998) did landscapes, from the tropics and warm and cool not say so, there is the strong likelihood that his temperate zones to the arid regions, encompassing eucalypts were mycorrhizal. Mycorrhizal plants rainforest, woodlands and pastoral country, have access N as free amino acids and, as well, are able acacias as a component of their understoreys or, to absorb N from proteins and chitins; see several somewhat less frequently, as dominant species. It references cited by Boddey et al. (2000b). Th ese follows, therefore, that acacias are widely considered latter sources of N also are probably absorbed in the as suitable species for revegetation of lands that form of amino acid which has been made available have become degraded due to inappropriate land by hydrolysis by mycorrhizal proteinases, chitinases use, salinity, erosion, waterlogging and other factors and other enzymes (cf. Leake and Read 1989, 1990). leading to loss of biodiversity. It follows also that, because of the wide diversity and distribution of the We submit that, in forest ecosystems, underground genus, there is almost invariably one or more Acacia transfer of N from N-fi xing trees to non-leguminous species appropriate for any particular revegetation trees is mediated by mycorrhizal pathways. Th is situation, with the possible exception of highly process seems more likely to be of particular saline soils. signifi cance early in the development of the ecosystem before other processes of N dynamics Acacia planting has been recognised as an become fully active. eff ective tool for erosion control since the early 1960s because, in general, acacias can be readily established on degraded land. At that time, the land reclamation: principles choice of species was most often determined merely and practice — the australian by seed availability, readiness of germination and experience tolerance of adverse environmental conditions, with Th is section is based largely on recent, mainly little concern for other principles of acclimation. unpublished experiences of Alison Jeavons and An unfortunate consequence was that some of Meigan Waayers and colleagues in the North the most easily established, vigorous species, e.g. Central Catchment Management Authority and A. baileyana and A. longifolia, became serious weeds the Department of Primary Industries, Victoria. outside their natural range. Nowadays, revegetation Th e observations have emerged from a series of practitioners try to establish as many species as

82 nitrogen fixation in acacias practicable, with special emphasis on the use of local relates to practicality. Outplanting tube stock grown species and provenances of species, especially in in a nursery into the fi eld is convenient and ideal for undertakings directed at restoration of biodiversity. a small number of seedlings. Indeed, outplanting is What precisely should be regarded as ‘local’ is the preferred option for getting greatest value from hotly debated. Th ere is a vocal body of opinion limited seed supplies. However, once the area to that specifi es that seed should be sourced no more be sown exceeds 2–3 hectares, economies of scale than 5 km from the intended point of revegetation. favour direct seeding.⁴ To give a general example, A much less restrictive defi nition is that any species the North Central Native Vegetation Plan of the is suitable for revegetation provided that it is not North Central Catchment Management Authority grown outside its natural range and that its seed is estimates that approximately 12,000 hectares of obtained from sites with the same soil type as the revegetation needs to be undertaken every year for revegetation site and with similar rainfall. Research the next 20 years in order to reverse the regional on these and related matters is currently in progress decline in biodiversity. Outplanting is clearly (A.G. Young, pers. comm.). not feasible for so vast an undertaking. A more specifi c example relates to a revegetation project for salinity control carried out on the Big Hill range General practice south of Bendigo, Victoria. Th is project saw the Th ere are two distinct methods of establishing establishment of about one million trees and shrubs acacias in revegetation undertakings: outplanting across nearly 1000 hectares of high recharge hills. tube stock and direct seeding. Each has its advantages and disadvantages, summarised in Table ⁴ Custom-built equipment for direct seeding has been 14. Th e major diff erence between the methods described by Dalton (1990) and Scheltema (1992). rall Peter Th Peter

Th e retention of vegetation, including species of Acacia, Eucalyptus and Allocasuarina, preserves the integrity of streamlines.

83 nitrogen fixation in acacias

Th e bulk of the work was carried out through direct been attempted by outplanting nursery-grown tube seeding, for two months in each of two years, and stock, it would have required the services of 65 involved three or four people. Th e cost of labour and people for fi ve days each week for four months, and materials was less than $100,000. Had the project the total cost would have exceeded $2,000,000.

Th e banks of this ephemeral watercourse have been seriously eroded following removal of vegetation. Further erosion can be readily contained by revegetating with surface-sown native species including acacias. rall Peter Th Peter Th is creek side, near St Arnaud, Victoria, was at risk because understorey shrubs had disappeared. It has recently

Alsion Jeavonsbeen revegetated by surface-sowing with native Botanic Gardens National of the Australian Couretsy species, Th ree-year-old roadside revegetation with acacias established including acacias inoculated with eff ective strains of root- by direct seeding nodule bacteria.

84 nitrogen fixation in acacias

Table 14. Comparison of the advantages and disadvantages of (i) direct seeding and (ii) outplanting of nursery-grown tube stock as means for establishment of acacias and other Australian native trees and shrubs to reclaim degraded land

Direct seeding Outplanting of nursery-grown tube stock Substantially lower costs than for other methods of Time tested for reliable results planting High densities of established plants are attainable Technology is well known and accepted Random plant distribution is attainable where a Appropriate where there is a need for fi xed plant natural ‘look’ is required density, uniform spacing or a particular species in a particular place Mature plants are more wind stable — because root Always slightly ahead in early growth compared with

Advantages systems have never been disturbed direct seeding; therefore useful when a quick eff ect is needed When using a wide range of species, savings on Supplements direct seeding where gaps occur in the propagation and planting far outweigh costs of plant stand additional seed

Direct seeding Outplanting of nursery-grown tube stock Low rainfall, seasonal variation, erodable or heavy clay Considerably higher costs and much more labour soil, insect predation and weed competition are all intensive, especially on a broad scale, than for direct more critical for direct seeding than for outplanting seeding Sometimes seedling establishment is patchy and gaps Only feasible for small-scale projects (probably never need fi lling exceeding 5 ha) Greater quantities of seed required than for nursery- grown seedlings Disadvantages Limited to species that germinate readily from (treated) seed Source: derived from Dalton (1990).

85 nitrogen fixation in acacias rall Courtesy of the Australian National BotanicCourtesy Gardens National of the Australian Peter Th Peter Th e integrity of this erosion-prone hillside on the Eyre Th e use of a knockdown herbicide allows surface-seeded Peninsula, South Australia, has been preserved by a stand of acacias to establish without competition from weeds. Acacia pycnantha (golden wattle). Bob Gault John Brockwell John Germination of acacia seed sown into the fi eld by direct Using direct seeding to establish salt-tolerant acacias on seeding is enhanced by application of smoked water into the salinity-endangered land at Bald Rock, Victoria. Th e seeder seed furrow. Th is picture, showing seed and water delivered shown here is a modifi cation of the one described by Dalton on to a concrete surface, is a simulation of the procedure. (1990).

86 nitrogen fixation in acacias

General considerations except following exposure to fi re, it tends to germinate slowly, intermittently and over a Availability of acacia seed long period. Often only 10–40% of viable seed A signifi cant constraint to the success of direct establishes beyond the seedling stage (Dalton 1990). seeding is seed availability. As a rule, acacia seed is Eff ective establishment, for both direct seeding easily collected from the wild. However, with many and tube stock, requires seed treatment to enhance species, the quantities of seed required for direct germinability. Immersion in boiling water (the seed seeding of degraded land (up to 0.5 kg per hectare) imbibes while the water is cooling) and mechanical often greatly outstrip the amounts available from scarifi cation are effi cient means for dealing with good quality remnant stands without drastic the problem of hard seed. Th e use of smoked depletion of resources necessary for sustaining water, either as a supplementary seed treatment populations of wildlife, especially birds. Th is is a or delivered directly into the seedbed alongside the particular problem in those circumstances where seed, further enhances germination. A butenolide local provenances are considered essential for has been identifi ed as the compound in smoked eff ective revegetation. As an example, the North water that stimulates seed germination (Flematti Central Catchment Management Authority has a et al. 2004). requirement for its revegetation program of 6000 kg acacia seed per year. Although the Authority has an Miscellaneous constraints to acacia seedling exceptionally good group of seed collectors, they establishment manage to collect only about 500 kg seed per year from within the catchment. Th e mining industry Naturally, seedling establishment of direct-seeded also has a large demand for seed for restoration acacias is best under good seasonal conditions. of mine sites where seeding rates may be up to Nonetheless, good results have sometimes been 35 kg per hectare. All this is refl ected in the cost of achieved during drought. commercially available acacia seed — $120–$510 per kg (Australian Seed Company 2003). Certain Even with good germination following seed Australian organisations with responsibility for treatment, early growth of direct-seeded acacias land reclamation are now tackling these problems is relatively slow and its continued success is through extension and training programs, one result dependent on eff ective control of fi rst-year being the establishment in recent years of a number competition from annual weeds. It is standard of acacia seed orchards. practice to seed directly into a furrow placed centrally along a strip of land from which weeds have been eliminated with a knock-down herbicide. Seed germination In addition to their competitive eff ect, some Acacia seed usually has high viability but is annual weeds harbour insect pests, e.g. red-legged notoriously hard-seeded. Under natural conditions, earth mite Halotydeus destructor (Tuck.) (Acarina:

87 nitrogen fixation in acacias

Eurodidae) on capeweed (Arctotheca calendula), early growth may well assist acacia seedlings to which feed on acacia seedlings. outrun fi rst-year competition from weeds. Th is is an especially important consideration in revegetating Herbivores, in particular rabbits, are partial to acacia riparian zones which, in Australia, are among the seedlings. Wherever large rabbit populations exist most endangered ecosystems. A further benefi t of in the vicinity of revegetation sites they must be rapid early growth is that it is likely to make acacia controlled . seedlings more tolerant of grazing by macropods (wallabies, kangaroos) and/or feral animals (rabbits, hares). Th e role of the acacia symbiosis in land reclamation Application of inoculant to acacia seed by seed Many reclamation projects involving acacias are coating is a time-consuming process, especially aimed at degraded land that once carried acacias where several seed lots require inoculation, and may as an understorey component or sometimes as the constitute a bottleneck at seeding time. A possible dominant species. Th e soils of such land, almost alternative is a free-fl owing granular inoculant certainly once contained populations of acacia (cf. Scudder 1975; Brockwell et al. 1980). Th is rhizobia which nodulated and fi xed N with their product, applied directly into the seed bed through hosts. However, with the disappearance of the a hopper attached to the seeding equipment, has acacias, it appears that their rhizobia have also been been used successfully in the United States for lost from the soil (Th rall et al. 2001b). Restoration inoculation of soybean and peanut crops (Scudder of healthy acacia communities therefore involves 1975). A similar device might be useful for direct the reintroduction of eff ective rhizobia as well as seeding of acacias. the acacias themselves. Recent research work (Th rall et al. 2005) has shown that rhizobial inoculation It seems reasonable to conclude that proper (by seed coating) of direct-seeded acacias more exploitation of the symbiosis might reduce the than doubles their establishment rate and often cost of acacia establishment in revegetation signifi cantly increases seedling growth rates. undertakings. Although exact costs are not yet known, it is estimated that a 50% reduction in An immediate consequence of these fi ndings on seed usage, combined with application of granular seedling establishment is that it becomes possible to inoculant directly into the seed bed, would reduce reduce direct seeding rates by at least half, reducing costs by at least 20–25%. costs and the demand for seed. Moreover, increased

88 7. General conclusions and prognosis

Th e body of literature relating to the symbiosis N fi xation in the genus Acacia, and the procedures between the genus Acacia and its diverse rhizobia for studying them, are generally little diff erent from that we have reviewed is dispersed in terms of the those of other, more extensively studied legume species investigated, the aspects of the symbiotic genera. Accordingly, where there appeared to be relationship studied and the places of publication no information about particular aspects of acacia of results. Th ere appear to be three main reasons symbiosis, we drew upon experience with other N- for this: fi rst, the genus is so large — some 1350 fi xing plants. species; second, it is more diffi cult to experiment with shrubs and trees than it is with herbs; third, the plant it has been unfashionable until recently to work with N-fi xing trees. With respect to the third Acacia is one of the largest genera of fl owering reason, the publication of newsletters such as NFT plants, widely distributed mainly in Australia, News: Improvement and Culture of Nitrogen Fixing Africa and Asia and with a multitude of products Trees (published by IUFRO and CSIRO Forestry extensively utilised by humans and wildlife. Th e and Forest Products, Canberra) and Farm Forestry genus is adapted to growth on many types of soil, News (once published by the Forestry/Fuelwood including those of low fertility, and is tolerant of Research and Development (F/FRED) Project, c/o arid and semi-arid environments. It is a prominent Winrock International: Arlington, VA — publication component of many forest ecosystems and, as a N- now ceased) has created forums for exchange of fi xing legume, apparently contributes substantially experience, views and ideas and has done much to natural N cycling. Although millions of hectares to overcome any resistance that might once have of land have been planted with acacias, especially for existed against working with N-fi xing trees. As the plantation and farm forestry and rehabilitation of reference list shows, the period since 1990 has seen degraded landscapes, many authorities consider that a proliferation of research and extension papers the genus is underutilised. Th e literature that we dealing with aspects of the symbiosis of acacias. surveyed gave us the very strong impression that the symbiosis of acacias with root-nodule bacteria, and It became clear to us during the review of the their capacity to fi x atmospheric N as a consequence literature that the processes of root nodulation and of that association, is also underutilised.

89 nitrogen fixation in acacias the bacteria important example of an acacia that appears highly Th e rhizobia that form root nodules on acacias specifi c is A. mangium. Th is species is native to appear to be at least as widely distributed as the northern Australia and Papua-New Guinea where plants themselves. Indeed, rhizobia capable of it apparently nodulates and fi xes N with resident nodulating and fi xing N with species of Acacia strains of rhizobia. Where it has been introduced can often be found in soils in which no acacias are into the tropics of the northern hemisphere, its N growing. Such organisms associate with legumes fi xation often appears to be impaired by the absence that are symbiotically related to Acacia species. of specifi c rhizobia. Th ere is a small taxonomic group of Acacia that is known to be non-nodulating. Otherwise, reports of the symbiosis failures of nodulation in natural ecosystems should be regarded cautiously. Lack of nodulation is more Th ere are numerous reports of measurements of likely to be due to harsh environmental conditions N fi xation by Acacia species. Several mensuration than to absence of rhizobia from the soil. procedures were used and the data that emerged are extremely variable — from virtually zero up Th ere are diverse groups of acacia rhizobia, including to 200 kg per hectare per annum. Th ere is no fast-growing, slow-growing and very-slow-growing suggestion that the variability was an artifact types. Organisms that form nodules on various of method, but some of the diff erences may acacias are certainly represented in at least four of have been a consequence of poorly eff ective or the currently recognised genera of rhizobia. Th is ineff ective symbioses. Some of the reports allowed number may be further expanded as the systematics a comparison of the amounts of N fi xed by acacias of the Rhizobaceae becomes clearer. and by other N-fi xing trees. Generally, Acacia fared poorly compared with genera such as Calliandria, It is inevitable with such diverse symbionts Gliricidia and Leucaena. An examination of the that host/bacterial relationships should exhibit circumstances revealed that the two data sets complex interactions leading to specifi city in both were not strictly comparable. Most measurements bacterium and plant in terms of nodule formation of acacia N fi xation had been made in natural and N fi xation. While there are very many strains ecosystems whereas the N fi xed by other tree of rhizobia that are able to nodulate many species legumes had been measured in anthropogenic of Acacia, the symbioses are often ineff ective or ecosystems. Th ere is a wealth of literature reporting poorly eff ective in fi xing N. In other words, many substantial diff erences in the N dynamics of the but not all Acacia species have a requirement for two systems. In natural ecosystems, N cycling takes specifi c rhizobial strains in order to express their place at a relatively rapid rate aided by diverse capacity to fi x atmospheric N. Th is characteristic macro- and microfl ora that decompose leaf litter has implications for the selection of strains of above ground and root fragments and non-living rhizobia for preparation of acacia inoculants. An components of the soil community underground.

90 nitrogen fixation in acacias

Th e organic N released by these processes is exploitation transformed to nitrate by nitrifying organisms. It Most of the world’s acacias grow in forests or is well known that nitrate is an inhibitor of legume woodlands. For reasons of microbial ecological nodulation and N fi xation. Where natural forest has competition with populations of acacia rhizobia been cleared for agriculture or silviculture, N cycling already resident in those soils, the chances of is much reduced, with lower rates of nitrifi cation successfully and permanently introducing more- and lower levels of soil nitrate. In the circumstances eff ective strains are nil. So there is probably little of these anthropogenic ecosystems, it is only that can be done to increase the N fi xation by acacias to be expected that legume N fi xation would be in natural ecosystems, except to improve their inhibited less than in natural ecosystems. We have vigour. It is a rule of thumb (Peoples et al. 1995a, b), concluded from these considerations, and despite perhaps somewhat oversimplifi ed, that the greater the superfi cial evidence to the contrary, that there a legume’s biomass the more N it fi xes. Applying is good reason to believe that the intrinsic N-fi xing this principle, correction of nutrient defi ciencies capacity of acacias is just as great as for other tree and control of insect pests, for instance, are legumes. legitimate means of increasing the vigour of acacias and, therefore, the amount of their atmospheric Th e diff erent elements of naturally occurring N fi xation. Economic considerations would populations of acacia rhizobia were extremely determine the practicalities of such an approach. variable in their N-fi xing eff ectiveness, even in Th ere is one circumstance where such a course association with Acacia species that grew in the of action merits consideration. Following timber immediate vicinity. Some highly eff ective strains harvesting or wildfi re in ecosystems where they have been isolated that showed their superiority occur, acacias are frequently the primary recolonising in glasshouse tests. Th ere were very few reports species and may be dominant for many months or of strain eff ectiveness trials in the fi eld. Th e most even several years. Any strategy that might be used compelling of these were the works of Galiana and at this time to increase their N fi xation would be of colleagues (Galiana et al. 1990, 1994, 1996, 1998) subsequent benefi t to non-N-fi xing components of in West Africa. Th ey demonstrated impressive the ecosystem. An appropriate strategy would also growth responses to inoculation of A. mangium with assist in the replenishment of the total pool of soil eff ective strains of rhizobia, as well as longevity N which occurs when the quantity of N removed as of the inoculant in the fi eld. Th e inoculation plant (or animal) product is less than the amount of was performed by sowing seed into nursery soil fi xed atmospheric N that remains behind in legume augmented with rhizobial culture. Th e seedlings residues. that emerged were well nodulated and continued to fi x N when they were outplanted into the fi eld. Where acacias are used in plantation and farm Th e procedure was termed inoculation by soil forestry, there is a great untapped potential for enrichment and has been exploited by others. exploiting the symbiosis. Nearly all plantation and

91 nitrogen fixation in acacias farm trees (and shrubs) are outplanted as nursery Th is is not a problem in most parts of the world, stock. When soil enrichment inoculation is applied where manufacture of mixed-strain legume to nursery soil immediately before seeding, the inoculants is very often standard practice. In large population of eff ective inoculant rhizobia in Australia, however, where there is a policy of single- the root medium leads to prompt nodulation of the strain inoculants, it would be necessary to have young acacia seedlings and early onset of N fi xation. packets of inoculant containing several individual Seedlings that have been inoculated reach planting culture packs and for the user to mix the contents size more quickly than uninoculated seedlings and, immediately before nursery soil inoculation by soil therefore, require less time in the nursery. When enrichment. Because demand for acacia inoculant the vigorous seedlings that result are outplanted is likely to be quite limited, it might be a problem into the fi eld, their survival and early growth are to fi nd manufacturers willing to produce small better than those of uninoculated seedlings. Th ese quantities of the specialised inoculant. Th e extent advantages may persist for some time and may lead of demand can be illustrated by a simple calculation. to early closure of canopy and lower maintenance At a conservatively sensible rate of soil enrichment costs. In addition, the eff ective inoculant strains inoculation, one kilogram of inoculant is enough persist in the soil for several years where they would for 10,000 acacia seedlings. Th is in turn is suffi cient remain a continuing potent source of inoculant for for 10 hectares of plantation. Th us, the total the infection and nodulation of new roots. requirement for a large planting of 1000 hectares is just 100 kg of inoculant. Th e very low cost of soil Th e principles applying to successful inoculation enrichment inoculation of nursery-grown acacia (Figure 3) suggested that peat culture, because it tube stock (a fraction of a cent each, which seems a appears to have a rather slower rate of mortality in small price to pay for increased vigour, survival and storage than liquid culture, may be the preferred growth of seedlings outplanted into the fi eld), may form of acacia inoculant for use in commercial be a two-edged sword. One means of dealing with nurseries. Indeed, peat inoculant is already the problem would be for nurseries to maintain their used in Australian plant nurseries that produce own stocks of acacia inoculant. Well-nodulated, large numbers of acacia seedlings as tube stock N-fi xing, diverse species of Acacia could be grown (Brockwell et al. 1999a). It was clear, for reasons together in an ‘inoculant’ plot, the soil from which of host/rhizobia specifi city, that a single strain of could later be used to enrich soil intended for the rhizobia could never be expected to nodulate and production of tube stock. Unfortunately for this fi x N with the complete range of Acacia species in proposal, some authorities legislate against the demand for plantation and farm forestry. Th us, it use of non-sterile soil in plant nurseries. A rather will be necessary to produce acacia inoculant that unsatisfactory solution to the problem suggested comprises several strains that, between them, are by Masutha et al. (1997) was to use only those (few) eff ective in N fi xation for the range of Acacia species species of Acacia that are suffi ciently promiscuous raised in nurseries. in their requirements for eff ective rhizobia that

92 nitrogen fixation in acacias they are likely to nodulate vigorously and fi x N would augment resources of soil N. For many abundantly with whatever populations of rhizobia species this will involve inoculation with eff ective occur naturally in soil. rhizobia. Th ere may also be scope, as suggested by Smith and Daft (1978), for dual inoculation with As a result of our literature review, we are convinced rhizobia and mycorrhizal fungi to remove other that there is considerable potential worldwide and nutritional constraints. Even in circumstances especially in Africa, Asia and Australia to enhance where inoculation is not practicable and/or feasible, the vigour, N-fi xing capacity and productivity of the the cultivation of acacias has potential to enhance many Acacia species by more fully and effi ciently soil fertility and soil structure in plantations, on exploiting their symbioses with root-nodule farms and in land-rehabilitation projects. bacteria. Simultaneously, improved symbioses Alison Jeavons Acacia mearnsii

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125

Appendix

In the text and tables, (i) acacias have been referred mentioned, with the authorities for those names. to by their botanical names (genus and species) Th e addition of the authority allows the plants to be whereas (ii) other plants have been referred to identifi ed unambiguously. It is customary to italicise by their botanical names and (where known) botanical names but not authorities. Common their common names. Mostly for acacias and names are also given. Th ere is no ‘correct’ common sometimes for other plants, the generic name has name for a plant. Where more than one common been abbreviated to an initial. Table A1 gives the name is given, the one listed fi rst is merely our own botanical names of all the plants that we have preference.

Table A1. Botanical and common namesa referred to in this review. Th e fi rst common name listed is the preferred common name. Botanical name and authority Common name(s) Acacias Acacia abyssinica Benth Not knowna Acacia acuminata Benth. Raspberry jam, jam wattle Acacia alata R. Br. Winged wattle Acacia albida Del.b Applering acacia, ana tree, winter thorn Acacia ampliceps Maslin Salt wattle Acacia aneura F. Muell ex Benth. Mulga Acacia arabica (Lam.) Willd.c Prickly acacia, babul Acacia ataxancantha DC. Not known Acacia aulacocarpa A. Cunn. ex Benth. Brush ironbark, hickory wattle Acacia auriculiformis A. Cunn. ex Benth. Earpod wattle Acacia bahiensis Benth. Not known Acacia baileyana F. Muell. Cootamundra wattle Acacia berlandieri Benth. Berlandia acacia, guajillo Acacia brevispica Harms Not known Acacia cambagei R.T. Baker Gidgee, stinking wattle Acacia catechu (L.f.) Willd. Catechu, black cutch Acacia caven (Molina) Molina Espino caven

127 nitrogen fixation in acacias

Table A1. (cont’d) Botanical and common namesa referred to in this review. Th e fi rst common name listed is the preferred common name. Botanical name and authority Common name(s) Acacias (cont’d) Acacia cincinnata F. Muell. Scorpion wattle Acacia confusa Merr. Not known Acacia crassicarpa A. Cunn. ex Benth. Lancewood Acacia cyanophylla Lindl.d Golden wreath wattle Acacia cyclops A. Cunn. ex G. Don Western coastal wattle Acacia dealbata Link Silver wattle Acacia decurrens Willd. Green wattle, early black wattle Acacia diffi cilis Maiden Not known Acacia elata A. Cunn. ex Benth. Mountain cedar wattle, cedar wattle Acacia erioloba E. Meyer Not known Acacia excelsa Benth. Ironwood, rosewood, ironwood wattle Acacia farnesiana (L.) Willd. Mimosa bush, sweet wattle Acacia fl eckii Schinz. Not known Acacia genistifolia Link Spreading wattle, early wattle Acacia gerrardii Benth. Not known Acacia glomerosa Benth. Not known Acacia greggii A. Gray Catclaw acacia, Texas mimosa Acacia harpophylla F. Muell. ex Benth. Brigalow Acacia hebeclada DC. Not known Acacia hereroensis Engl. Not known Acacia hilliana Maiden Not known Acacia holocericea A. Cunn. ex G. Don Candelabra wattle Acacia homalophylla A. Cunn. ex Benth. Yarran Acacia imbricata F. Muell. Imbricate wattle Acacia implexa Benth. Lightwood, hickory wattle Acacia irrorata Sieber ex Spreng. Green wattle Acacia julifera Benth. Not known Acacia karroo Hayne Karroo thorn Acacia kempeana F. Muell. Witchetty bush Acacia kirkii Oliver Not known Acacia koa A. Gray Koa acacia, koa Acacia leptocarpa A. Cunn. ex Benth. Not known Acacia leucophloea (Roxb.) Willd. Nimbar Acacia leucophylla Lindl. Not known

128 nitrogen fixation in acacias

Table A1. (cont’d) Botanical and common namesa referred to in this review. Th e fi rst common name listed is the preferred common name. Botanical name and authority Common name(s) Acacias (cont’d) Acacia littorea Maslin Western Australian coastal dune wattle Acacia longifolia (Andrews) Willd. Sydney golden wattle, sallow wattle Acacia macrostachya DC. Not known Acacia maidenii F. Muell. Maiden’s wattle Acacia mangium Willd. Brown salwood, hickory wattle Acacia martii Benth. Not known Acacia mearnsii De Wild. Black wattle, green wattle Acacia melanoxylon R. Br. Blackwood, Tasmanian blackwood Acacia mellifera (M. Vahl.) Benth. Not known Acacia mucronata Willd. ex H.L. Wendl. Narrow-leaved wattle, variable sally Acacia neriifolia A. Cunn. ex Benth. Silver wattle, oleander wattle Acacia nigrescens Oliver Knob thorn Acacia nilotica (L.) Del. Prickly acacia, babul Acacia notabilis F. Muell. Hickory wattle Acacia oxycedrus Sieber ex DC. Spike wattle Acacia papyrocarpa Benth. Western myall Acacia paradoxa DC. Kangaroo thorn Acacia parramattensis Tindale Parramatta wattle, green wattle Acacia pellita O. Schwarz. Not known Acacia pendula A. Cunn. ex G. Don Weeping myall, boree, myall Acacia pennata (L.) Willd. Not known Acacia penninervis Sieber ex DC Mountain hickory Acacia pentogona (Schum.) Hook. f. Not known Acacia peuce F. Muell. Waddywood Acacia plectocarpa A. Cunn. ex Benth. Not known Acacia podalyriifolia A. Cunn. ex G. Don Queensland silver wattle, Mt Morgan wattle Acacia polyacantha Willd. Not known Acacia polyphylla DC. Not known Acacia pulchella R. Br. Prickly Moses Acacia pycnantha Benth. Golden wattle, broad-leaved wattle Acacia raddiana Savie Umbrella thorn Acacia redolens Maslin Ongerup wattle Acacia refi ciens Wawra Not known Acacia retinodes Schldl. Wiralda, swamp wattle

129 nitrogen fixation in acacias

Table A1. (cont’d) Botanical and common namesa referred to in this review. Th e fi rst common name listed is the preferred common name. Botanical name and authority Common name(s) Acacias (cont’d) Acacia salicina Lindley Cooba, native willow, doolan Acacia saligna (Labill.) Wendl.d Golden wreath wattle Acacia schweinfurthii Brenan & Exell Not known Acacia senegal (L.) Willd. Gum arabic, kher, senegal gum Acacia seyal Del. Shittimwood, talh, thirty-thorn Acacia signata F. Meull. Not known Acacia silvestris Tind. Bodalla silver wattle, red wattle Acacia smallii Iselyf Huisache Acacia stenophylla A. Cunn. ex Benth. Eumong, river cooba Acacia suaveolens (Smith) Willd. Sweet wattle, sweet-scented wattle Acacia terminalis (Salisb.) J.F. Macbr. Sunshine wattle, New Year wattle Acacia tetragonophylla F. Muell. Dead fi nish, kurara Acacia tortilis (Forsskal) Haynee Umbrella thorn Acacia trachycarpa Pritz. Sweet-scented minnie-ritchie Acacia trachyphloia Tind. Golden feather wattle Acacia tumida F. Muell. ex Benth. Pindan wattle Acacia vernicifl ua Cunn. Varnish(ed) wattle, manna wattle Acacia victoriae Benth. Prickly wattle, elegant wattle, gundabluie Faidherbia albida (Del.) A. Chev.b Applering acacia, ana tree, winter thorn Other leguminous trees Aotus ericoides (Vent.) G. Don Not known Cassia siamea Lam.g Djoowar, kassod-tree, Siamese senna Cassia spectabilis DC.g Not known Chamaecytisus proliferus (L.f.) Link Tree lucerne Leucaena esculenta (Mocino & Sesse ex DC.) Benth. Not known Leucaena leucocephala (Lam.) De Wit Leucaena, ipil-ipil, leadtree, jumbie bean Mimosa affi nis Harms ex Glaz. Not known Parkia biglobosa (Jacq.) R. Br. ex G. Don African locust bean, nitta Prosopis chilensis (Molina) Stuntz Algarrobo, Chilean algarrobo Senna siamea (Lam.) H. Irwin & Barnebyg Djoowar, kassod-tree, Siamese senna Senna spectabilis (DC.) H. Irwin & Barnebyg Not known Tamarindus indica L. Tamarind

130 nitrogen fixation in acacias

Table A1. (cont’d) Botanical and common namesa referred to in this review. Th e fi rst common name listed is the preferred common name. Botanical name and authority Common name(s) Other legumes Abrus precatorius L. Not known Aeschynomene indica L. Kat sola Amorpha fruticosa L. False indigo, indigo bush, bastard indigo Amphicarpaea trisperma Baker Not known Arachis hypogea L. Peanut, groundnut, goober, mani Aspalathus carnosa P.J. Bergius Not known Astragalus adsurgens Pallas Not known Astragalus sinicus L. Chinese milk vetch Cajanus cajan (L.) Millsp. Pigeonpea Coronilla varia L.h Crown vetch Cicer arietinum L. Chickpea, garbanzo Daviesia ulicifolia C.R.P. Andrews Gorse bitter pea Desmodium intortum (Miller) Fawc. & Rendle Greenleaf desmodium Desmodium sinuatum Blume ex Baker Not known Galega offi cinalis L. Goats-rue, galega Galega orientalis Lam. Not known Glycine max (L.) Merr. Soybean Glycine soja Siebold & Zucc. Wild soybean Glycine wightii (Wight & Arn.) Verdc.i Perennial glycine, creeping glycine Gueldenstaedtia multifl ora Bunge Not known Hardenbergia violacea (Schneev.) Stearn False sarsaparilla Kennedia prostrata R. Br. Running postman Lotononis bainesii Baker Lotononis Medicago polymorpha L. Common burr medic, California bur clover Medicago ruthenica (L.) Trautv. Not known Medicago sativa L. Lucerne, alfalfa Neonotonia wightii (Wight & Arn.) Lackeyi Perennial glycine, creeping glycine Neptunia natans (Willd.) W. Th eobald Not known Phaseolus vulgaris L. French bean, kidney bean, navy bean Pisum sativum L. Pea, fi eld pea Securigera varia (L.) Lassenh Crown vetch Sesbania herbacea (Mill.) R. McVaugh Not known Sesbania rostrata Bremek. & Oberm. Not known Trifolium alexandrinum L. Berseem clover, berseem

131 nitrogen fixation in acacias

Table A1. (cont’d) Botanical and common namesa referred to in this review. Th e fi rst common name listed is the preferred common name. Botanical name and authority Common name(s) Other legumes (cont’d) Trifolium ambiguum M. Bieb. Caucasian clover, kura clover, honey clover Trifolium dubium Sibth. Suckling clover Trifolium fragiferum L. Strawberry clover Trifolium glomeratum L. Cluster clover, ball clover Trifolium hybridum L. Alsike clover Trifolium incarnatum L. Crimson clover Trifolium pratense L. Red clover, cowgrass Trifolium repens L. White clover Trifolium subterraneum L. Subterranean clover Vigna umbellata (Th unb.) Ohwi & H.Ohashi Ricebean Vigna unguiculata (L.) Walp. Cowpea Non-legumes Allium cepa L. Onion Arctotheca calendula (L.) Levyns Capeweed Cucos mucifera L. Coconut Eucalyptus globulus Labill. Tasmanian blue gum Eucalyptus nitens (Deane & Maiden) Maiden Shining gum Lactuca sativa L. Lettuce Olax phillanthi R. Br. Mistletoe Parasponia andersonii Planch. Not known Sorghum bicolor (L.) Moench s. lat. Sorghum, sweet sorghum, broom millet Th eobroma cacao L. Cocoa Zea mays L. Maize, corn a Authorities and common names taken from Hartley (1979), National Academy of Sciences 1979, Leigh et al. 1981, Simmons (1981, 1988), Brooker and Kleinig (1990), Wiersema et al. (1990), Tame (1992), Lazarides and Hince 1993, Searle (1996), Boxshall and Jenkyn (2001a, 2001b, 2001c, 2001d), Orchard and Wilson (2001a, 2001b), ILDIS 2002, IPNI 2002, Australian Seed Co. (2003) and Kutsche and Lay (2003). Sometimes ILDIS (2002) gives more than one authority for a particular plant species; in such cases, we give the most recent. ‘Not known’ refers to species for which none of the above gives a common name. b Acacia albida is obsolete; the species is now known as Faidherbia albida. c Acacia arabica is obsolete; the species is now known as A. nilotica subsp. nilotica. d Acacia cyanophylla is obsolete; the species is now known as A. saligna. e Acacia raddiana is obsolete; the species is now known as A. tortilis subsp. raddiana. f Acacaia smallii is obselete; the species is now known as A. farnesiana var. farnesiana. g Senna siamea and S. spectabilis were formerly Cassia siamea and C. spectabilis, respectively, now obselete. h Coronilla varia is obsolete; the species is now known as Securiga varia. i Glycine wightii is obsolete; the species is now known as Neonotonia wightii.

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